Objective To investigate the genetic diversity of sandfly populations in endemic areas of visceral leishmaniasis in China, so as to provide references insights into management of visceral leishmaniasis and the vector sandflies. MethodsSixteen sampling sites were selected from main endemic foci of visceral leishmaniasis in China from June to September 2024, including Shanxi Province, Shaanxi Province, Henan Province, Gansu Province, Sichuan Province, and Xinjiang Uygur Autonomous Region. Sandflies were captured using light traps and manual aspirators from sheep pens, chicken coops, cave dwellings, bovinesheds, and pig pens at each sampling site. A single sandfly sample was washed in phosphate-buffered saline (PBS), and genomic DNA was extracted from sandfly samples. Cytochrome oxidase subunit 1 (COI) gene was amplified using PCR assay with universal primers, and analyzed and retrieved with the nucleotide sequence analysis tool (BLAST) software, and the sequence of COI gene was aligned with the ClustalX 1.83 and MEGA 7.0 software. The base composition and variation site of the COI gene sequence were analyzed using the software MEGA 7.0, and the number of haplotypes, total number of segregating sites, haplotype diversity, nucleotide diversity, and average nucleotide differences were calculated in the COI gene sequence using the software DnaSP 5.10, followed by Tajima’s D test for neutrality. Haplotypes were screened using the software DnaSP 5.10, and the haplotype network map of sandfly samples was plotted using the software Network 5.0. MEGA 7.0 software was employed for gene sequence editing and alignment, and calculation of genetic distances among sandfly species sampled from different regions, and a phylogenetic tree was built with a neighbor-joining method. Results A total of 466 sandflies were captured from 16 sampling sites in China from June to September 2024, and 430 gene sequences were yielded following PCR amplification and sequencing of the COI gene, with 652 to 688 bp in the length of amplification fragments. The captured sandfly samples were characterized as Phlebotomus chinensis, Sergentomyia squamirostris, Se. koloshanensis, Ph. sichuanensis, and Ph. longiductus following the COI gene sequence alignment in BLAST. A total of 251 haplotypes were identified in the 430 gene sequences from sandfly samples (50.5%), and the average haplotype diversity, nucleotide diversity and average number of nucleotide difference were 0.885, 0.257 and 160.761, respectively. The Tajima’s D values were -0.92 for sandfly populations from Yangquan City, Shanxi Province and -1.73 for sandfly populations from Sanmenxia City, Henan Province, and were all more than 0 for sandfly populations from other sampling sites. Haplotype analysis identified 50 haplotypes, which were classified into two haplogroups. Heplogroup 1 included 29 haplotypes, which had a high homology, and heplogroup 2 included 21 haplotypes. The average genetic distance was 0.000 to 0.604 among sandfly samples from different sampling sites, and phylogenetic analysis revealed that the five sandfly species were clustered into distinct clades, all with 100% clade confidence. Conclusions There is a high genetic polymorphism in the COI gene from five sandfly populations in main endemic foci of visceral leishmaniasis in China, and COI gene may serve as a marker gene for analysis of the genetic structure of sandfly populations.

Objective:To investigate the differences and applicability of free silica detection methods of different crystal forms in dust, and to provide a basis for the selection of various methods.Methods:From December 2021 to June 2022, dust samples from 20 enterprises in different industries in 18 cities in Henan Province were randomly selected as the investigation objects. X-ray diffraction (XRD) method was used to analyze the samples and classify the samples. Based on GBZ/T 192.4-2007 "Determination of Dust in the Air of Workplace-Part 4: Content of Free Silica in Dust", pyrophosphate method and infrared spectrophotometry were used for quantitative determination. The measured results were analyzed by paired sample t test to evaluate the advantages and disadvantages of the two methods and their applicable scope. Results:The XRD results of 20 dust samples could be divided into α, β, γ crystal types and the mixed type of α and γ. There was no significant difference between pyrophosphate method and infrared spectrophotometry ( P=0.180). The pyrophosphate method results of β, γ and α, γ mixed crystalline free silica were significantly higher than those of infrared spectrophotometry, and the difference was statistically significant ( P<0.001) . Conclusion:Pyrophosphate method and infrared spectrophotometry are suitable for α-type free silica, while pyrophosphate method is suitable for β, γ and α, γ mixed crystalline free silica.

Objective:To investigate the differences and applicability of free silica detection methods of different crystal forms in dust, and to provide a basis for the selection of various methods.Methods:From December 2021 to June 2022, dust samples from 20 enterprises in different industries in 18 cities in Henan Province were randomly selected as the investigation objects. X-ray diffraction (XRD) method was used to analyze the samples and classify the samples. Based on GBZ/T 192.4-2007 "Determination of Dust in the Air of Workplace-Part 4: Content of Free Silica in Dust", pyrophosphate method and infrared spectrophotometry were used for quantitative determination. The measured results were analyzed by paired sample t test to evaluate the advantages and disadvantages of the two methods and their applicable scope. Results:The XRD results of 20 dust samples could be divided into α, β, γ crystal types and the mixed type of α and γ. There was no significant difference between pyrophosphate method and infrared spectrophotometry ( P=0.180). The pyrophosphate method results of β, γ and α, γ mixed crystalline free silica were significantly higher than those of infrared spectrophotometry, and the difference was statistically significant ( P<0.001) . Conclusion:Pyrophosphate method and infrared spectrophotometry are suitable for α-type free silica, while pyrophosphate method is suitable for β, γ and α, γ mixed crystalline free silica.

Objective:To observe the effect of acupuncture combined with vitamin B12 point injection on pain severity in patients with discogenic low back pain and to analyze its potential mechanisms. Methods:A total of 96 patients with discogenic low back pain were randomly divided into two groups.The control group received acupuncture treatment,while the combined group received vitamin B12 point injection in addition to the identical acupuncture treatment in the control group.Both groups were treated for 2 weeks.The visual analog scale(VAS)and Oswestry disability index(ODI)scores were compared before treatment,after 1 week of treatment,and after 2 weeks of treatment.The levels of such serum inflammatory factors as tumor necrosis factor(TNF)-α and interleukin(IL)-6,serum beta-endorphin(β-EP),and prostaglandin(PG)E2 were compared before and after treatment.Adverse reactions and clinical efficacy were compared between the two groups after treatment. Results:The total effective rate in the combined group was higher than that in the control group(P<0.05).The VAS and ODI scores in the combined group after 1 week and 2 weeks of treatment were lower than those in the control group at the same time point(P<0.05).The levels of TNF-α,IL-6,and PGE2 in the combined group were lower than those in the control group after treatment(P<0.05),while the level of β-EP was higher than that in the control group(P<0.05).There was no statistical difference in the overall incidence of adverse reactions between the two groups(P>0.05). Conclusion:Acupuncture combined with vitamin B12 point injection can alleviate pain and promote functional recovery in patients with discogenic low back pain;reducing the levels of TNF-α,IL-6,and PGE2 and increasing the level of β-EP may be part of the mechanism of the therapy.

Objective To investigate the effect and mechanism of Nuanxinkang on plaque formation in obstructive sleep apnea-hypopnea syndrome(OSAHS)comorbid with atherosclerosis(AS)mice by inhibiting endothelial-to-mesenchymal transition(EndMT).Methods Male ApoE-/-mice were randomly divided into six groups:control group,model group,atorvastatin group(2.6 mg·kg-1)and Nuanxinkang low-,medium-and high-dose groups(crude drug 3.5,7.0,14.0 g·kg-1),with eight mice in each group.The mice were exposed to chronic intermittent hypoxia(CIH)environment during sleep for a long time,and fed with high-fat diet to replicate OSAHS comorbid with AS mouse model.Oil red O staining was used to observe the formation of plaque on aortic intima in mice.Masson trichrome staining was used to evaluate the collagen content of atherosclerotic plaques in the aortic root of mice.The expressions of endothelial cell marker CD31 and EndMT marker Vimentin in aortic plaque were detected by immunofluorescence.Blood lipid levels were determined by ELISA;the mRNA expression levels of EndMT markers α-SMA and Cdh2 in aortic tissue were detected by qPCR.Results Compared with the control group,the area of aortic atherosclerotic plaque in the model group was significantly increased(P＜0.01),and the area of collagen deposition in the aortic root plaque was significantly increased(P＜0.01).The number of CD31 positive cells in the plaque were significantly decreased(P＜0.01),and the number of Vimentin positive cells were significantly increased(P＜0.01).Serum TG,T-CHO and LDL-C levels were significantly increased(P＜0.01),and HDL-C level was significantly decreased(P＜0.01).The mRNA expression levels of α-SMA and Cdh2 in aortic tissue were significantly increased(P＜0.01).Compared with the model group,the area of aortic atherosclerotic plaque in Nuanxinkang groups were significantly reduced(P＜0.05,P＜0.01),and the collagen deposition area of aortic root atherosclerotic plaque were significantly reduced(P＜0.05,P＜0.01).The number of CD31 positive expression cells in the plaque of Nuanxinkang high-dose group were significantly increased(P＜0.05),and the number of Vimentin positive expression cells in the plaque of Nuanxinkang medium-and high-dose groups were significantly decreased(P＜0.05,P＜0.01).The serum TG level of mice in the high-dose group of Nuanxinkang was significantly decreased(P＜0.01).The serum T-CHO and LDL-C levels of mice in each Nuanxinkang administration group were significantly decreased(P＜0.05,P＜0.01).The serum HDL-C levels of mice in the medium-and high-dose groups of Nuanxinkang were significantly increased(P＜0.01).The mRNA expression levels of α-SMA and Cdh2 in aortic tissue of mice in each treatment group were significantly decreased(P＜0.01).Conclusion Nuanxinkang can effectively reduce the plaque formation in OSAHS comorbid with atherosclerosis mice,which may be related to its inhibition of EndMT and reduction of collagen fiber formation.

Objective:To systematically review the risk prediction models for cognitive impairment in stroke patients, aiming to provide references for clinical healthcare professionals in selecting or constructing high-quality risk assessment tools.Methods:A computerized search was conducted in PubMed, Embase, Web of Science, OVID, Cochrane Library, SinoMed, CNKI, Wanfang Database, and VIP to identify studies related to risk prediction models for cognitive impairment in stroke patients. The search was limited to articles published up to August 1, 2023. Two researchers independently screened the literature, extracted data, and assessed the risk of bias and applicability of the included studies using PROBAST.Results:A total of 26 articles were included. The applicability of the studies was generally good, but all studies had some degree of bias risk, mainly arising from unreasonable study designs, inappropriate time intervals between predictor assessment and outcome determination, insufficient sample sizes, unreasonable handling of continuous variables, omission of missing data, lack of reporting of calibration, and overfitting of the models. Meta-analysis results showed that age ( OR=0.05, 95% CI: 0.033-0.057), education level ( OR=-0.13, 95% CI: -0.171 - -0.082), history of diabetes ( OR=2.32, 95% CI: 1.867-2.881), history of hypertension ( OR=0.67, 95% CI: 0.420-0.918), and NIHSS score ( OR=0.40, 95% CI: 0.331-0.469) were factors for cognitive impairment in stroke patients. Conclusions:While various risk prediction models for cognitive impairment in stroke patients exist, they suffer from methodological flaws and high bias risks, with some commonalities and controversies in predictors. Future research should adhere to the principles of transparent reporting of individual prognosis or diagnosis of multivariate prediction models, develop localized prediction models with low bias risk and good applicability, and conduct internal and external validations to demonstrate their applicability and feasibility in clinical practice.

In order to understand the prevalence and genetic variation of H9N2 subtype avian influ-enza virus in Shandong,a total 492 tracheal and lung tissue samples collected from chicken farms with respiratory symptoms in partial areas in Shandong were detected by H9 subtype AIV real-time RT-PCR,and the positive samples were inoculated with chicken embryos for two generations.Whole genome sequences of the positive strains by applying Illumina Miaseq platform,and genetic evolution and mutation at positions associating with viral pathogenicity and transmissibility were analyzed.The results showed that there were 72 samples were positive for H9 subtype AIV among the 492 samples,with a positive rate of 14.63％.Thirty-four strains of H9 subtype AIV were ob-tained from the positive samples after passing through chicken embryo,meanwhile,the 34 isolates were all H9N2 subtype AIV by whole genome sequencing analysis.By analyzing the evolutionary tree of HA and NA genes,HA and NA genes of the 34 H9N2 AIV strains belonged to Y280-like branch and F/98-like branch,respectively.Meanwhile,based on above branches,there were obvious time node subbranch,which one was"isolates before 2013",another one was"isolates after 2013".The HA cleavage sites of thirty-four H9N2 strains were all 325PSRSSR↓GLF333,which met the se-quence characteristics of the lowly pathogenic avian influenza virus,and the HA receptor binding site 226 amino acid was leucine,which had the characteristics of blinding to a-2,6 mammalian sialic acid receptors.Among the internal amino acid sites that are key to mammalian adaptation,all strains had an I368V mutation in the PB1 gene that enhanced viral transmissibility in mammals and the PB2 genes of some strains were mutated to enhance the mammalian adaptation of I292 V and A588 V.The above results illustrated that the H9N2 subtype AIV gene segments in Shandong have different degrees of recombination and gene variation,so it is necessary to strengthen the monito-ring of virus variation.

Objective:To analyze the impact of cecal ligation and puncture (CLP)-induced sepsis on the proliferation and differentiation of intestinal epithelial cells.Methods:① Animal experiment: sixteen male C57BL/6 mice were divided into sham operation group (Sham group) and CLP-induced sepsis model group (CLP group) by random number table method, with 8 mice in each group. After 5 days of operation, the jejunal tissues were taken for determination of leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) and intestinal alkaline phosphatase (IAP) by polymerase chain reaction (PCR). The translation of LGR5 was detected by Western blotting. The expression of proliferating cell nuclear antigen (Ki67) was analyzed by immunohistochemistry. IAP level was detected by modified calcium cobalt staining and colorimetry. Immunofluorescence staining was used to detect the expression of Paneth cell marker molecule lysozyme 1 (LYZ1) and goblet cell marker molecule mucin 2 (MUC2). ② Cell experiment: IEC6 cells in logarithmic growth stage were divided into blank control group and lipopolysaccharide (LPS) group (LPS 5 μg/mL). Twenty-four hours after treatment, PCR and Western blotting were used to analyze the transcription and translation of LGR5. The proliferation of IEC6 cells were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining. The transcription and translation of IAP were detected by PCR and colorimetric method respectively.Results:① Animal experiment: the immunohistochemical results showed that the positive rate of Ki67 staining in the jejunal tissue of CLP group was lower than that of Sham group [(41.7±2.5)% vs. (48.7±1.4)%, P = 0.01]. PCR and Western blotting results showed that there were no statistical differences in the mRNA and protein expressions of LGR5 in the jejunal tissue between the CLP group and Sham group (Lgr5 mRNA: 0.7±0.1 vs. 1.0±0.2, P = 0.11; LGR5/β-actin: 0.83±0.17 vs. 0.68±0.19, P = 0.24). The mRNA (0.4±0.1 vs. 1.0±0.1, P < 0.01) and protein (U/g: 47.3±6.0 vs. 73.1±15.3, P < 0.01) levels of IAP in the jejunal tissue were lower in CLP group. Immunofluorescence saining analysis showed that the expressions of LYZ1 and MUC2 in the CLP group were lower than those in the Sham group. ②Cell experiment: PCR and Western blotting results showed that there was no significant difference in the expression of LGR5 between the LPS group and the blank control group (Lgr5 mRNA: 0.9±0.1 vs. 1.0±0.2, P = 0.33; LGR5/β-actin: 0.71±0.18 vs. 0.69±0.04, P = 0.81). The proliferation rate of IEC6 cells in the LPS group was lower than that in the blank control group, but there was no significant difference [positivity rate of EdU: (40.5±3.8)% vs. (46.5±3.6)%, P = 0.11]. The mRNA (0.5±0.1 vs. 1.0±0.2, P < 0.01) and protein (U/g: 15.0±4.0 vs. 41.2±10.4, P < 0.01) of IAP in the LPS group were lower than those in the blank control group. Conclusion:CLP-induced sepsis inhibits the proliferation and differentiation of intestinal epithelial cells, impairing the self-renewal ability of intestinal epithelium.

Objective To investigate the effect and molecular mechanism of safflower yellow(SY)on wound healing of diabetic foot ulcer(DFU)in mice.Methods Forty-five C57BL/6 mice were injected intraperitoneally with streptozotocin to establish diabetic models.The diabetic mice were randomly divided into the sham operation group,model group,low-dose SY intervention group,high-dose SY intervention group,and high-dose SY combined with insulin-like growth factor-1(IGF-1)group,with 9 mice in each group.Before modelling,mice in the model group were not given any intervention,mice in the low-dose SY intervention group and high-dose SY intervention group were injected intraperitoneally with 5 and 20 mg·kg-1 SY,respectively,and mice in the high-dose SY combined with IGF-1 group were injected intraperitoneally with 20 mg·kg-1 SY and 0.03 mg·kg-1 IGF-1.Except for the sham operation group,the DFU model was established by incising the dorsal skin of the foot in the remaining four groups of mice.No wound on the dorsal skin of the foot was made in the sham operation group,and the remaining surgical steps were the same as those in the model group.The body mass of mice in each group was measured on day 14 after modelling using an electronic scale,tail vein blood was collected for fasting blood glucose measurement,and the wound width was measured using a small vernier caliper.Then,the mice were executed to collect the wound tissues.Hematoxylin-eosin staining was used to detect the histopathological changes in the wound tissues of mice in each group.Reverse transcription-quantitative real-time polymerase chain reaction was used to measure the relative expression levels of platelet-derived growth factor(PDGF),vascular endothelial growth factor(VEGF),alpha-smooth muscle actin(α-SMA),type Ⅰ collagen(collagen Ⅰ),protein tyrosine phosphatase 1B(PTP1B)and advanced glycation end products(AGEs)mRNA in the wound tissues of mice in each group.Western blot was used to detect the relative expression levels of proliferation marker Ki-67,proliferating cell nuclear antigen(PCNA),apoptosis-associated proteins(caspase-3,caspase-6,and caspase-7),p85 phosphatidylinositol-3-kinase(PI3K)and phosphorylated protein kinase B(p-AKT)protein in the wound tissues of mice in each group.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-6 in the wound tissues of mice in each group.Results The differences in blood glucose and body mass of mice among the sham operation group,model group,low-dose SY intervention group,high-dose SY intervention group,and high-dose SY combined with IGF-1 group were not statistically significant(P＞0.05).The healing rate of wound tissues in the high-dose SY intervention group was significantly greater than that in the model group,low-dose SY intervention group,and high-dose SY combined with IGF-1 group(P＜0.01).There was no statistically significant difference in the healing rate of wound tissues among the model group,low-dose SY intervention group,and high-dose SY combined with IGF-1 group(P＞0.05).In the high-dose SY intervention group,a large number of collagen fibers were densely and orderly arranged in the wound tissues,accompanied by a large number of neovessels;in the model group,low-dose SY intervention group,and high-dose SY combined with IGF-1 group,the wound tissues were sparsely populated with collagen fibers,accompanied by a small number of neovessels.The relative expression levels of PDGF,VEGF,α-SMA and collagen Ⅰ mRNA in the wound tissues of mice in the high-dose SY intervention group were significantly higher than those in the model group,low-dose SY intervention group,and high-dose SY combined with IGF-1 group(P＜0.01);the relative expression levels of PTP1B and AGEs mRNA in the wound tissues of mice in the high-dose SY intervention group were significantly lower than those in the model group,low-dose SY intervention group,and high-dose SY combined with IGF-1 group(P＜0.01).The relative expression levels of PDGF,VEGF,α-SMA,collagen Ⅰ,PTP1B and AGEs mRNA showed no statistically significant difference among the model,low-dose SY intervention,and high-dose SY combined with IGF-1 groups(P＞0.05).The relative expression levels of Ki-67 and PCNA protein in the wound tissues of mice in the high-dose SY intervention group were significantly higher than those in the model group,low-dose SY intervention group,and high-dose SY combined with IGF-1 group(P＜0.01);the relative expre-ssion levels of caspase-3,caspase-6,caspase-7,p85 PI3K,and p-AKT protein were significantly lower than those in the model group,low-dose SY intervention group,and high-dose SY combined with IGF-1 group(P＜0.01).There was no statistically significant difference in the relative expression levels of Ki-67,PCNA,caspase-3,caspase-6,caspase-7,p85 PI3K and p-AKT protein among the model,low-dose SY intervention,and high-dose SY combined with IGF-1 groups(P＞0.05).The levels of TNF-α,IL-1β and IL-6 in the wound tissues of mice in the high-dose SY intervention group were significantly lower than those in the model,low-dose SY intervention,and high-dose SY combined with IGF-1 groups(P＜0.01).There was no statistically significant difference in the levels of TNF-α,IL-1β and IL-6 in the wound tissues of mice in the model,low-dose SY intervention and high-dose SY combined with IGF-1 groups(P＞0.05).Conclusion High-dose SY intervention promotes DFU wound healing in mice by increasing angiogenesis,collagen formation and cell proliferation and reducing insulin resistance,inflammatory response and cell apoptosis,which may be related to the inhibition of the PI3K/AKT pathway.

Objective To investigate the effects of eicosanoic acid(C20DC)on the proliferation and migration of human retinal endothelial cells(HRECs)and its mechanism.Methods The optimal working concentration of C20DC in human retinal pigment epithelium 19(ARPE-19)cells and HRECs was determined as 30 mg·L-1 and 25 mg·L-1,respec-tively.HRECs were divided into the C20DC treatment group(HRECs treated with C20DC)and the control group[HRECs treated with dimethyl sulfoxide(DMSO)].The effects of C20DC on the migration and proliferation of HRECs were detec-ted by cell proliferation and migration experiments.The molecular docking method was used to simulate the binding ability of C20DC to peroxisome proliferator-activated receptor δ(PPARδ).ARPE-19 cells were divided into the C20DC+ARPE-19 group(ARPE-19 cells treated with C20DC)and the DMSO+ARPE-19 group(ARPE-19 cells treated with DMSO).The ex-pression levels of PPARδ and angiopoietin-like protein 4(ANGPTL4)in ARPE-19 cells and ANGPTL4 protein in HRECs were detected using Western blot.The ANGPTL4 protein expression levels in ARPE-19 cells and HRECs were quantitatively analyzed using enzyme-linked immunosorbent assay(ELISA).Results Compared with the control group,the prolifera-tion and migration of cells in the C20DC treatment group significantly increased(both P＜0.05),and C20DC could stably bind to PPAR8(binding energy:-7.20 kcal·mol-1).Western blot showed that the expression level of ANGPTL4 protein in the C20DC+ARPE-19 group was higher than that in the DMSO+ARPE-19 group,and the difference was statistically sig-nificant(P＜0.05);there was no statistically significant difference in the expression level of PPARδ receptor protein be-tween the two groups(P＞0.05).The expression level of ANGPTL4 protein in the C20DC treatment group was higher than that in the control group,and the difference was statistically significant(P＜0.05).ELISA quantitative analysis showed that the expression level of ANGPTL4 in the C20DC+ARPE-19 group was higher than that in the DMSO+ARPE-19 group(P＜0.001);the expression level of ANGPTL4 in the C20DC treatment group was higher than that in the control group,and the difference was statistically significant(P＜0.05).Conclusion C20DC can promote the expression of ANGPTL4 pro-tein by binding to PPARδ and thus increase the proliferation and migration of retinal related cells(HRECs and ARPE-19 cells).Its mechanism may be related to the increased angiogenesis in retinopathy of prematurity.