Based on the interleukin-17(IL-17) signaling pathway, this study explores the effect and mechanism of Bufei Decoction on Klebsiella pneumoniae pneumonia in rats. SD rats were randomly divided into the control group, model group, Bufei Decoction low-dose group(6.68 g·kg~(-1)·d~(-1)), Bufei Decoction high-dose group(13.36 g·kg~(-1)·d~(-1)), and dexamethasone group(1.04 mg·kg~(-1)·d~(-1)), with 10 rats in each group. A pneumonia model was established by tracheal drip injection of K. pneumoniae. After successful model establishment, the improvement in lung tissue damage was observed following drug administration. Core targets and signaling pathways were screened using transcriptomics techniques. Real-time fluorescence quantitative polymerase chain reaction was used to detect the mRNA expression of core targets interleukin-6(IL-6), interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and chemokine CXC ligand 6(CXCL6). Western blot was used to assess key proteins in the IL-17 signaling pathway, including interleukin-17A(IL-17A), nuclear transcription factor-κB activator 1(Act1), tumor necrosis factor receptor-associated factor 6(TRAF6), and downstream phosphorylated p38 mitogen-activated protein kinase(p-p38 MAPK), and phosphorylated nuclear factor-κB p65(p-NF-κB p65). Apoptosis of lung tissue cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL). The results showed that, compared with the control group, the model group exhibited significant pathological damage in lung tissue. The mRNA expression of IL-6, IL-1β, TNF-α, and CXCL6, as well as the protein levels of IL-17A, Act1, TRAF6, p-p38 MAPK/p38 MAPK, and p-NF-κB p65/NF-κB p65, were significantly increased, and the number of apoptotic cells was notably higher, indicating successful model establishment. Compared with the model group, both low-and high-dose groups of Bufei Decoction showed reduced pathological damage in lung tissue. The mRNA expression levels of IL-6, IL-1β, TNF-α, and CXCL6, and the protein levels of IL-17A, Act1, TRAF6, p-p38 MAPK/p38 MAPK, and p-NF-κB p65/NF-κB p65, were significantly decreased, with a significant reduction in apoptotic cells in the high-dose group. In conclusion, Bufei Decoction can effectively improve lung tissue damage and reduce inflammation in rats with K. pneumoniae. The mechanism may involve the regulation of the IL-17 signaling pathway and the reduction of apoptosis.
Animals ; Interleukin-17/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Rats ; Male ; Klebsiella pneumoniae/physiology* ; Klebsiella Infections/immunology* ; Humans ; Lung/drug effects*

OBJECTIVE: By analyzing the differential miRNA in seminal plasma between individuals with normal and abnormal sperm DNA fragmentation index（DFI）, we aim to identify miRNA that may impact sperm DNA integrity and target genes, and attempt to analyze their potential mechanisms of action. METHODS: A total of 161 study subjects were collected and divided into normal control group, DFI-medium group and DFI-abnormal group based on the DFI detection values. Differential miRNA were identified through miRNA chip analysis. Through bioinformatics analysis and target gene prediction, miRNA related to DFI and specific target genes were identified. The relative expression levels of differential miRNA and target genes in each group were compared to explore the impact of their differential expression on DFI. RESULTS: Through miRNA chip analysis, a total of 11 differential miRNA were detected. Bioinformatics analysis suggested that miR-26a-5p may be associated with reduced sperm DNA integrity. And gene prediction indicated that PTEN was a specific target gene of miR-26a-5p. Compared to the normal control group, the relative expression levels of miR-26a-5p in both the DFI-medium group and the DFI-abnormal group showed a decrease, while the relative expression levels of PTEN showed an increase. The relative expression levels of miR-26a-5p in all groups were negatively correlated with DFI values, while the relative expression levels of PTEN showed a positive correlation with DFI values in the DFI-medium group and the DFI-abnormal group. The AUC of miR-26a-5p in the DFI-medium group was 0.740 (P<0.05), with a sensitivity of 73.6% and a specificity of 71.5%; the AUC of PTEN was 0.797 (P<0.05), with a sensitivity of 76.5% and a specificity of 78.4%. In the DFI-abnormal group, the AUC of miR-26a-5p was 0.848 (P<0.05), with a sensitivity of 81.3% and a specificity of 78.1%. While the AUC of PTEN was 0.763 (P<0.05), with a sensitivity of 77.2% and a specificity of 80.2%. CONCLUSION miR-26a-5p affects the integrity of sperm DNA by regulating the expression of PTEN negatively. The relative expression levels of seminal plasma miR-26a-5p and PTEN have good diagnostic value for sperm DNA integrity damage, which can help in the etiological diagnosis and prognosis analysis of abnormal DFI. This provides a diagnostic and treatment approach for the study and diagnosis of DFI abnormalities without clear etiology.
Male ; Humans ; MicroRNAs/genetics* ; PTEN Phosphohydrolase/genetics* ; Spermatozoa ; Semen/metabolism* ; DNA Fragmentation

OBJECTIVE: To investigate the inhibitory effect of Tanreqing Injection (TRQ) on the activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome in macrophages infected with influenza A virus and the underlying mechanism based on mitophagy pathway. METHODS: The inflammatory model of murine macrophage J774A.1 induced by influenza A virus [strain A/Puerto Rico/8/1934 (H1N1), PR8] was constructed and treated by TRQ, while the mitochondria-targeted antioxidant Mito-TEMPO and autophagy specific inhibitor 3-methyladenine (3-MA) were used as controls to intensively study the anti-inflammatory mechanism of TRQ based on mitophagy-mitochondrial reactive oxygen species (mtROS)-NLRP3 inflammasome pathway. The levels of NLRP3, Caspase-1 p20, microtubule-associated protein 1 light chain 3 II (LC3II) and P62 proteins were measured by Western blot. The release of interleukin-1β (IL-1β) was tested by enzyme linked immunosorbent assay, the mtROS level was detected by flow cytometry, and the immunofluorescence and co-localization of LC3 and mitochondria were observed under confocal laser scanning microscopy. RESULTS: Similar to the effect of Mito-TEMPO and contrary to the results of 3-MA treatment, TRQ could significantly reduce the expressions of NLRP3, Caspase-1 p20, and autophagy adaptor P62, promote the expression of autophagy marker LC3II, enhance the mitochondrial fluorescence intensity, and inhibit the release of mtROS and IL-1β (all P<0.01). Moreover, LC3 was co-localized with mitochondria, confirming the type of mitophagy. CONCLUSION TRQ could reduce the level of mtROS by promoting mitophagy in macrophages infected with influenza A virus, thus inhibiting the activation of NLRP3 inflammasome and the release of IL-1β, and attenuating the inflammatory response.
Mitophagy/drug effects* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Animals ; Macrophages/virology* ; Inflammasomes/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Mitochondria/metabolism* ; Reactive Oxygen Species/metabolism* ; Influenza A virus/physiology* ; Interleukin-1beta/metabolism* ; Cell Line ; Injections

Objective To investigate the effect of sanguinarine(SAN)on proliferation and ferroptosis of colorectal cancer cells.Methods SW620 and HCT-116 cells treated with different concentrations of SAN were examined for cell viability changes using CCK8 assay to determine the IC50 of SAN in the two cells.The inhibitory effects of SAN on proliferation,invasion and migration of the cells were evaluated using colony-forming assay and Transwell assays.ROS production in the treated cells was analyzed with flow cytometry,and lipid peroxide production was assessed by detecting malondialdehyde(MDA)level.Glutathione(GSH)levels in the cells were detected,and Western blotting was used to detect the expressions of ferroptosis-related proteins STUB1 and GPX4.Results SAN significantly inhibited the proliferation,invasion and migration of SW620 and HCT-116 cells.SAN treatment significantly promoted ROS production,increased intracellular MDA level,and lowered GSH level in the two cells(P<0.05).Western blotting showed that SAN significantly upregulated the expression of STUB1 and down-regulated the expression of its downstream protein GPX4(P<0.05).Conclusion SAN induces ferroptosis in colorectal cancer cells by regulating STUB1/GPX4,which may serve as a new therapeutic target for colorectal cancer.

Objective To investigate the effect of sanguinarine(SAN)on proliferation and ferroptosis of colorectal cancer cells.Methods SW620 and HCT-116 cells treated with different concentrations of SAN were examined for cell viability changes using CCK8 assay to determine the IC50 of SAN in the two cells.The inhibitory effects of SAN on proliferation,invasion and migration of the cells were evaluated using colony-forming assay and Transwell assays.ROS production in the treated cells was analyzed with flow cytometry,and lipid peroxide production was assessed by detecting malondialdehyde(MDA)level.Glutathione(GSH)levels in the cells were detected,and Western blotting was used to detect the expressions of ferroptosis-related proteins STUB1 and GPX4.Results SAN significantly inhibited the proliferation,invasion and migration of SW620 and HCT-116 cells.SAN treatment significantly promoted ROS production,increased intracellular MDA level,and lowered GSH level in the two cells(P<0.05).Western blotting showed that SAN significantly upregulated the expression of STUB1 and down-regulated the expression of its downstream protein GPX4(P<0.05).Conclusion SAN induces ferroptosis in colorectal cancer cells by regulating STUB1/GPX4,which may serve as a new therapeutic target for colorectal cancer.

Background::Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.Methods::The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 +) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. Results::The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10-unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10-unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. Conclusions::This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Objective:To clone the Helicobacter pylori(Hp)hp0169 gene and conduct the crystallographic study,and to clarify its secondary and tertiary structures.Methods:The hp0169 gene and its encoded protein sequence of the Hp NCTC26695 strain were retrieved from the UniProt database.Bioinformatics method was used to analyze the physicochemical properties of the Hp recombinant protease(HpPrtC)protein;SOPMA and DNAStrar softwares were used to predict the secondary structure characteristics of HpPrtC protein;SWISS-MODEL software was used to construct the tertiary structure of the HpPrtC protein;IEDB and ABCpred softwares were used to predict the antigenic epitopes of the B lymphocytes HpPrtC protein;SYFPEITMI website was used to predict the antigenic epitopes of the T lymphocytes of HpPrtC protein;the expert pool(EP)and random forest(RF)algorithms were used to predict the crystallizability of the HpPrtC protein;the HpPrtC recombinant protein was expressed in the prokaryotic system;the HpPrtC recombinant protein was purified by Ni2+affinity chromatography and size-exclusion chromatography;the crystallization conditions for HpPrtC were screened by crystallization kit.Results:The hp0169 gene contained 1 269 base pairs and encoded the protein of 422 amino acids,the theoretical isoelectric point was 7.64 and the relative molecular weight was 47 300.HpPrtC was a hydrophilic and soluble protein.The number of amino acids of alpha helices of HpPrtC accounted for 35.78%,beta sheets 18.72%,beta turns 6.87%,and random coils 38.63%.The antigen epitope analysis results showed that HpPrtC contained five dominant linear epitopes of B lymphocytes,three conformational epitopes,and multiple potential dominant epitopes of T lymphocytes.The homology modeling results showed that HpPrtC formed a dimer,and each monomer displayed a barrel structure surrounded by β sheets,alpha helices,and random coils.HpPrtC was predicted to have moderate crystallizability without signal peptides and transmembrane helices.Small clustered needle-like crystals of HpPrtC were obtained under the conditions of 0.2 mol·L-1 magnesium chloride,0.1 mol·L-1 tris(hydroxymethyl)amino methane(Tris),3.4 mol·L-1 hexanediol,and pH=8.5.Conclusion:HpPrtC is a hydrophilic protein that forms a dimeric structure and crystallizes into small clustered needle-like crystals under suitable conditions.HpPrtC contains dominant antigenic epitopes of the T lymphocytes and B lymphocytes and can serve as an antigen for the design of Hp vaccines to establish the multivalent fusion vaccines or multi-epitope vaccines;the results provide an experimental basis for the prevention and control of Hp.

Objective To investigate the correlation between serum levels of prostaglandin-endoperoxide synthase 2(PTGS2),chitinase-3-like protein 1(CHI3L1)and cognitive impairment caused by cerebrovascular disease.Methods From October 2020 to October 2022,96 inpatients with cerebrovascular diseases admitted to the Third People's Hospital of Chengdu were regarded as the study subjects.The basic clinical data of the patients were recorded,the serum levels of PTGS2 and CHI3L1 were detected by enzyme-linked immunosorbent assay,and these patients were grouped into normal group(n=60)and impaired group(n=36)based on the presence or absence of cognitive impairment.The correlation between serum PTGS2 and CHI3L1 levels and fasting blood glucose(FBG)and homocysteine(Hcy)was analyzed by Pearson method.Logistic regression model was used to determine whether serum PTGS2 and CHI3L1 were independent risk factors for predicting cognitive impairment.Receiver operating characteristic(ROC)curve was drawn,and the predictive value of CHI3L1 and serum PTGS2 expression level in cognitive impairment in patients with cerebrovascular disease was analyzed according to the area under the curve(AUC).Results Compared with the normal group,the levels of serum PTGS2(29.30±9.46 pg/ml vs 17.86±5.40 pg/ml)and CHI3L1(13.04±4.06 pg/ml vs 7.51±2.66 pg/ml)in the disorder group were increased,and the differences were statistically significant(t=7.553,8.065,all P＜0.05).Multivariate Logistic regression analysis showed that FBG(OR=3.612,95%CI:2.324～5.614),Hcy(OR=2.584,95%CI:1.351～4.944),PTGS2(OR=1.964,95%CI:1.194～3.231)and CHI3L1(OR= 1.556,95%CI:1.023～2.367)were independent risk factors of cognitive impairment(all P＜0.05).PTGS2 was positively correlated with FBG and Hcy(r=0.368,0.551,all P＜0.05),and CHI3L1 was positively correlated with FBG and Hcy(r=0.510,0.376,all P＜0.05).The ROC curve showed that the area under curve(AUC)of PTGS2 and CHI3L1 in predicting cognitive impairment was 0.819 and 0.829,respectively.The AUC of the combined prediction of cognitive impairment was 0.902,which was obviously higher than that of the independent prediction of the two(Z =2.089,2.293;P=0.037,0.021),with sensitivity and specificity of 77.78%and 98.33%,respectively.Conclusion PTGS2 and CHI3L1 were highly expressed in the serum of patients with cognitive impairment of cerebrovascular disease,indicating that both were related to cognitive impairment of patients with cerebrovascular disease.

Objective:To investigate the efficacy of double S-shaped elastic stable intramedullary nailing in the treatment of paediatric fractures of the distal tibia diaphyseal metaphyseal junction.Methods:From January 2018 to January 2022, a total of 25 children with fracture of the distal tibia diaphyseal metaphyseal junction were treated at Department of Pediatric Orthopedics, The Second Affiliated Hospital of Inner Mongolia Medical University. All of them were treated with closed reduction and double S-shaped elastic stable intramedullary nailing. There were 16 males and 9 females with an average age of (10.4±3.3) years, and 14 left sides and 11 right sides. The operation time, imaging results and complications were recorded after operation. At the last follow-up, the American Orthopaedic Foot & Ankle Society (AOFAS) scoring was used to evaluate the efficacy.Results:Closed reduction succeeded in all patients. The operation time was (55.6±23.7) min. Follow-up lasted (20.5±4.7) months for this cohort. Bony union was achieved in all patients after (11.5±2.7) weeks. No postoperative complications occurred in the patients, like infection, loss of reduction, disparity in length of lower limbs, delayed union or non-union. The AOFAS scoring at the last follow-up yielded 23 excellent and 2 good cases, and an excellent and good rate of 100% (25/25).Conclusion:In the treatment of paediatric fractures of the distal tibia diaphyseal metaphyseal junction, double S-shaped elastic stable intramedullary nailing is a safe, effective and feasible option.

Objective To observe the effects and safety of semaglutide and saxagliptin combined with metformin in the treatment of type 2 diabetes mellitus(T2DM)with abdominal obesity(AO).Methods The data of patients with T2DM and AO were retrospectively analyzed.Patients with T2DM and AO were divided into semaglutide group and saxagliptin group according to the cohort method.Both groups were given metformin orally,0.5 g a time,tid.On this basis,the semaglutide group was injected with semaglutide,0.25 mg a time,2 weeks later,the dose was adjusted to 0.5 mg a time,qw.The saxagliptin group was given oral administration of saxagliptin on the basis of metformin,5 mg a time,qd.All patients received 3 months of treatment.Glucose metabolism indexes,pancreatic islet function indexes,adipokines and adverse drug reactions were compared between the groups.Results There were 48 cases in semaglutide group and 49 cases in saxagliptin group.After treatment,the levels of fasting plasma glucose(FPG)in the semaglutide group and the saxagliptin group were(7.45±1.22)and(7.98±1.30)mmol·L-1;the levels of 2 h plasma glucose(2 h PG)were(9.78±1.64)and(10.55±1.82)mmol·L-1;the levels of hemoglobin A1c(HbA1c)were(5.66±0.94)and(6.25±0.87)％;the levels of fasting insulin(FINS)were(29.12±2.46)and(34.34±7.15)pmol·L-1;the levels of fasting C-peptide(FC-P)were(292.66±67.53)and(319.03±77.92)pmol·L-1;homeostatic model assessment-insulin resistance(HOMA-IR)were 2.51±0.78 and 2.94±0.89;homeostatic model assessment-β(HOMA-β)were 51.74±15.31 and 43.72±14.56;levels of Irisin were(4.56±0.32)and(4.17±0.54)ng·L-1;the levels of spexin(SPX)were(0.71±0.15)and(0.64±0.17)ng·mL-1;the levels of asprosin(ASP)were(1.22±0.16)and(1.34±0.25)μg·L-1.The above indexes were significantly different between semaglutide group and saxagliptin group(all P＜0.05).The total incidence rates of adverse drug reactions in semaglutide group and saxagliptin group were 10.42％(5 cases/48 cases)and 2.04％(1 case/49 cases),without statistically significant difference(P＞0.05).Conclusion The combination of semaglutide and metformin can lower blood glucose and in patients with T2DM and AO more significantly,and improve pancreatic function and adipokines,with good safety.