abstract:
Objective To study the effect of sex-determining region Y-frame protein 3(SOX3) on proliferation andestradiol secretion in human ovarian granulosa cells(KGN cell line). Methods The gene sequence of human SOX3(NM＿005634.3) was searched in Gene-Bank, an NCBI database, and the target geneSOX3was amplified by PCR, which was cloned into lentiviral vector pLV-EF1a-GFP-2A-Puro to obtain the overexpression lentiviral recombinant plasmid pLV-EF1a-GFP-2A-Puro-SOX3; the correctly sequenced overexpressed lentiviral recombinant plasmid as well as packaging plasmids(pGag/Pol, pRev, pVSV-G) were co-transfected into human embryonic kidney cell line(HEK 293T) cells(pLV-SOX3 group), and pLV-EF1a-GFP-2A-Puro and packaging plasmids(pGag/Pol, pRev, pVSV-G) were co-transfected into HEK 293T cells(pLV-NC group), the lentiviral particles of both groups were collected and the titers of the viruses were measured after 48 h of transfection, the lentiviruses of the two groups were infected into KGN cells, and the stably expressed cell lines were obtained after puromycin screening for 2 weeks; real-time fluorescence quantitative PCR(RT-qPCR) and Western blot were used to detect the SOX3 mRNA and protein levels in the two groups; CCK-8 assay was used to detect the proliferative ability of the cells in the two groups; ELISA was used to determine the concentration of estradiol in the two groups. Results The identification of PCR products and sequencing results showed that theSOX3gene fragment was amplified successfully, and the enzyme digestion and sequencing results indicated that the construction of overexpression lentiviral recombinant plasmid was completed; green fluorescence could be detected after lentiviral infection of HEK 293T cells, which indicated that lentiviral packaging was successful; the lentivirus was screened by puromycin after lentiviral infection of KGN cells, and the cells were observed to express green fluorescence under the fluorescence microscope; RT-qPCR and Western blot assays both showed that the expression level of SOX3 in the pLV-SOX3 group was significantly higher than that in the pLV-NC group(P<0.05). CCK-8 assay results showed that the proliferation ability of the cells in the pLV-SOX3 group significantly increased compared with that in the pLV-NC group(P<0.01). ELISA results showed that estradiol concentration was elevated in the pLV-SOX3 group compared with the pLV-NC group(P<0.05). Conclusion Overexpression of the transcription factor SOX3 can promote the proliferation and estradiol secretion of human ovarian granulosa cells KGN.

abstract:
Objective Given that the PI3K/AKT/mTOR signaling pathway is associated with the progression of knee osteoarthritis(KOA), this study aims to investigate whether the polarization induction of synovial macrophages mediated by the PI3K/AKT/mTOR signaling axis is the cause of KOA progression. Methods The synovial fluid of KOA KL-Ⅱ and KL-Ⅲ patients and normal individuals was collected, and the percentage of M1 macrophages(CD80, CD86) and M2 macrophages(CD163, CD206) in the synovial fluid(M1/M2 ratio) was measured to evaluate the polarization of macrophage cytokines such as IL-1, IL-6, IL-10, and tumor necrosis factor(TNF)-α, transforming growth factor(TGF)-β Expression in KOA synovial fluid, and detect and analyze of key molecules PI3K/AKT/mTOR signaling axis PI3K, AKT3, mTORC1, and inducible nitric oxide synthase(iONS) in KOA synovial fluid. Results Compared with the synovial fluid of normal individuals, the percentage of M1 macrophages(CD80, CD86) in KOA patients increased(P<0.01), and the M1/M2 ratio increased(P<0.001); The expression of IL-1, IL-6, and TNF-α in the synovial fluid of the KOA group was also higher than that of the control group(P<0.01), while the expression of IL-10 and TGF-β in the KOA group was significantly reduced(P<0.01); The key proteins PI3K, AKT3, mTORC1, and downstream inflammatory factor iONS in the PI3K/AKT/mTOR signaling pathway in the synovial fluid of the KOA group were higher than those in the control group(P<0.01). Conclusion In KOA synovial fluid, M1 macrophage polarization plays a dominant role, and the inflammatory response mediated by M1 macrophage polarization may be the cause of synovitis. At the same time, the PI3K/AKT/mTOR signaling pathway may mediate the polarization of M1 macrophages involved in KOA inflammatory response.

abstract:
Objective To construct hepatocyte-specific silence information regulator 3(Sirt3) gene knockout(Sirt3Δhep) mice by Cre-loxP technique,and to provide an important animal model for further studying the biological function of the hepatocyte Sirt3 gene in diseases.Methods LoxP-labeled Sirt3flox/floxmice were mated with Alb-Cre homozygous(Alb-Cre+/+) mice,and the F1 generation Sirt3flox/-/Alb-Cre+/-mice were then mated with Sirt3flox/floxmice,and the F2 genotype of Sirt3flox/flox/Alb-Cre+/-mice were the Sirt3Δhepmice constructed in this experiment.Sirt3flox/flox/Alb-Cre-/-(Sirt3flox/flox) mice were the control mice.Mouse tail genome DNA was extracted and PCR was used to identify the genotypes of the offspring mice.Immunofluorescence was used to detect Sirt3 expression in mouse hepatocytes.Primary hepatocytes and tissue proteins of Sirt3Δhepmice were extracted,and the expression of Sirt3 in mouse hepatocytes and other tissues was verified by Western blot.HE staining was used to observe mice's liver,heart,spleen,and lung tissue structure.Results Sirt3Δhepmice were successfully identified.Immunofluorescence and Western blot results demonstrated a significant decrease in the expression of Sirt3 in the hepatocytes of these mice compared to the control group(P<0.01).At the same time,there was no significant difference in the expression of Sirt3 in the heart,spleen,kidney,and lung tissues of Sirt3Δhepmice compared with the control group(P>0.05).The results of HE staining showed that the histological characteristics of the liver,heart,spleen,lungs,kidneys,and other major organs of Sirt3Δhepmice were not significantly different from those of the control group mice.Conclusion Hepatocyte-specific Sirt3 gene knockout mice are successfully constructed,which provides an animal model to explore further the role and molecular mechanism of the hepatocyte Sirt3 gene in diseases.

abstract:
Objective To investigate the effects of sulforaphane(SFN) in regulating the macrophage glycolysisviathe arachidonate 5-lipoxygenase(ALOX5)/nuclear factor kappa B(NF-κB) signaling pathway on the progression of diabetic nephropathy(DN). Methods Bioinformatics analysis was used to identify the target genes of SFN in the treatment of DN. Human proximal tubular epithelial cell line(HK-2 cells) was induced with 30 mmol/L high glucose(HG) to create an in vitro model of DN. HK-2 cells were divided into the following groups: normal glucose(NG) group, HG group, HG+SFN(3 mmol/L) group, HG+ALOX5 group, HG+SFN(3 mmol/L) +ALOX5 group, HG-treated macrophages+HK-2 group, HG+SFN(3 mmol/L) treated macrophages s+HK-2 group, HG+ ALOX5 transfection treated macrophages+HK-2 group, HG+SFN(3 mmol/L) + ALOX5 transfection treated macrophages+HK-2 group. CCK-8 assay was used to detect cell viability, Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) method was used to detect cell apoptosis; glucose and lactate levels in the cells were measured using assay kits; Western blot was performed to detect the expression of ALOX5, NF-κB, and glycolysis-related proteins hexokinase-2(HK2), pyruvate kinase M2(PKM2), glucose transporter 1(GLUT1) in each group. Diabetic nephropathy(DN) mouse models were established using streptozotocin(STZ) and treated with SFN(0.5 mg/kg). Various biochemical parameters were measured in the mice, and kidney tissue pathology was examined using H&E staining. Western blot was used to detect the expression of glycolysis-related proteins(HK2, PKM2, GLUT1) in kidney macrophages. Results Bioinformatics analysis revealedALOX5as the target gene of SFN in treating DN. Compared to the HG group, SFN treatment enhanced HK-2 cell viability and inhibited apoptosis(P<0.05); concurrently, SFN treatment suppressed HG-induced macrophage glycolysis-related protein and attenuated macrophage-mediated HK-2 cellular injury(P<0.05). Western blot results showed that SFN inhibited the expression of ALOX5 and NF-κB(P<0.05). The mouse experiment results showed that SFN treatment improved kidney function and pathological changes in the kidney of DN mice, and inhibited the related protein expression of acrophage glycolysis in kidney tissue(P<0.05). Conclusion SFN improves the progression of DN by inhibiting the expression of macrophage glycolysis-related protein through theALOX5/NF-κB signaling pathway.

abstract:
Objective To construct lentiviral vector ofp62gene over-expression, and stably expressp62gene in human monocytic leukemia cells 1(THP-1), and to provide a way to study the role ofp62gene at the cellular level. Methods Thep62gene fragment was amplified by polymerase chain reaction(PCR), and the amplified product was ligated to the linearized pcDNA3.1-Flag-PCDH10 lentiviral vector. After identifying with PCR, the PCR product was cotransfected with the packaging plasmid into human embryonic kidney cells 293(HEK 293T). THP-1 cells were infected with recombinant lentivirus. Positive cell clones were screened by ampicillin. Western blot and real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) were used to detect THP-1 cell lines with highp62expression(overexpression group) and THP-1 cell lines transfected with empty plasmid withoutp62gene(control group). The expression levels of TNF-α, IL-1β and Cxcl1 afterK.p. infection were detected by RT-qPCR. Results Thep62gene fragment was successfully obtained by PCR and ligated to pcDNA3.1-Flag-PCDH10 vector. PCR confirmed thatp62-pcDNA3.1-Flag-PCDH10 recombinant plasmid was constructed successfully. Ampicillin-resistant cell lines were selected after lentiviral infection of THP-1 cells. The results of Western blot analysis showed that the THP-1 cells with drug sieve survival increased the expression of P62 protein compared with the control cells(P<0.001), and RT-qPCR analysis showed that the relative mRNA expression ofp62increased(P<0.001). THP-1 cells with high expression of P62 were successfully constructed. The levels of TNF-α、IL-1β and Cxcl1 from THP-1 cells with high expression of P62 significantly increased after infection withK.p.(P<0.01). Conclusion P62-pcDNA3.1-Flag-PCDH10 vector and THP-1 cells with high expression of P62 can be successfully constructed by three-plasmid packaging system, which provides a basis for the study ofp62.

abstract:
Objective To investigate the role and mechanism of circular RNA containing pleckstrin homology domain of Pleckstrin homology domain family M member 3(circRNA＿PLEKHM3) in regulating epithelial-mesenchymal transition(EMT) behavior in cervical cancer cells through the miR-320 and KLF4. Methods The expression levels of circRNA＿PLEKHM3 in cervical cancer cells Hela and CaSki were detected by real-time quantitative PCR(qRT-PCR). RNA fluorescence in situ hybridization was used to determine the localization of circRNA＿PLEKHM3 in human cervical cancer epithelial cells CaSki. Dual luciferase reporter gene experiments were conducted to investigate the targeting relationship betweencircRNA＿PLEKHM3andmiR-320, as well as the targeting relationship betweenmiR-320andKLF4. CaSki cells were overexpressed withcircRNA＿PLEKHM3. Additionally, three groups were set up: overexpression ofmiR-320on the basis ofcircRNA＿PLEKHM3overexpression, silencing ofKLF4on the basis ofcircRNA＿PLEKHM3overexpression, and silencing ofKLF4on the basis ofmiR-320overexpression. qRT-PCR was performed to detect the expression levels of miR-320 in CaSki. Western blot experiments were conducted to determine the expression of KLF4 and EMT markers including E-cadherin, N-cadherin, Vimentin, MMP-2, and MMP-9 in CaSki cells. Transwell assays were performed to measure cell migration and invasion. Results The expression of circRNA＿PLEKHM3 decreased in Hela and CaSki cells(P<0.05), mainly localized in the cytoplasm. The dual luciferase reporter gene experiment demonstrated a targeting relationship betweenmiR-320andcircRNA＿PLEKHM3, as well as betweenKLF4andmiR-320. Overexpression ofcircRNA＿PLEKHM3inhibited the protein expression of miR-320, N-cadherin, Vimentin, MMP-2, and MMP-9, up-regulated the protein expression of E-cadherin, and reduced cell migration and invasion(P<0.05). Overexpression ofmiR-320or silencing ofKLF4on the basis ofcircRNA＿PLEKHM3overexpression both promoted the protein expression of miR-320, N-cadherin, Vimentin, MMP-2, and MMP-9, down-regulated the protein expression of E-cadherin, and increased cell migration and invasion(P<0.05). However, silencing ofKLF4on the basis ofmiR-320overexpression inhibited the protein expression of KLF4, N-cadherin, Vimentin, MMP-2, and MMP-9, up-regulated the protein expression of E-cadherin, and reduced cell migration and invasion(P<0.05). Conclusion Overexpression ofcircRNA＿PLEKHM3regulates EMT in cervical cancer cells through the miR-320/KLF4 axis.

abstract:
Objective To construct myeloid-specificSpi1gene knockout mice and analyze their genotypes, so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets. Methods According to the principle of CRISPR/Cas9 technology and Cre/LoxP system, sgRNA and Donor vectors were designed and constructed. The transcript of Exon 2(Exon 2) was used as the knockout region, andLoxpelements were placed on both sides of Exon 2. Cas9 protein, sgRNA and Donor vector were mixed and microinjected into the fertilized eggs of C57BL/6J mice, the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice, and F0 generation was obtained after 19～20 days. Positive F0 mice were mated with C57BL/6J mice to obtain stable F1Spi1flox/+mice.Spi1flox/+mice of F1 generation were selfed to obtainSpi1flox/floxmice.Spi1flox/floxmated withLyz2-Cre+mice to obtainSpi1flox/+/Lyz2-Cre+mice, and then mated withSpi1flox/flox, theSpi1flox/flox/Lyz2-Cre+mice were myeloid-specificSpi1gene knockout(KO) mice.Spi1flox/flox/Lyz2-cre-mice were used as wild-type(WT)mice. DNA of WT and KO mice was extracted, and the genotypes were identified by agarose gel electrophoresis after PCR amplification. Western blot was used to detect the expression of spleen focus forming virus proviral integration oncogene, Spi-1/purine rich box-1(PU.1) in immune cells of WT and KO mice. Results The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer wasSpi1flox/floxhomozygote, and the genotype of mice with 700 bp amplified byLyz2-Cre primer wasLyz2-Cre+. Western blot showed that compared with WT group, the protein PU.1 was not expressed in bone marrow-derived macrophages(BMDMs) and peritoneal macrophages(PM) in KO group(P<0.01). There was no significant difference of statistics in the expression level of PU.1 in T cells between KO mice and WT mice. The results of PCR and Western blot showed that myeloid-specificSpi1KO mice were successfully constructed. Conclusion The myeloid-specificSpi1gene KO mice are successfully constructed and identified, which provides animal model basis for further revealing the potential mechanism of PU.1 inimmune regulation.

abstract:
Objective To investigate the expression of miR-27a in colorectal cancer cell, and to analyze the effect of its targeted regulation of(Secreted Frizzled-Related Protein, SFRP1) on the biological behavior of colorectal cancer cells. Methods Real-time fluorescent quantitative PCR(qRT-PCR) was employed to examine the expression of miR-27a and SFRP1 mRNA in colorectal cancer tissues and adjacent normal tissues. Western blot was used to detect the expression of SFRP1 protein in colorectal cancer tissues and adjacent normal tissues. TargetScan software and dual luciferase reporter gene test were used to detect the targeted regulation of miR-27a on SFRP1.HCT116 cells were transfected with miR-27a mimic, miR-27a inhibitor and negtive control(NC). The expression of miR-27a and SFRP1 mRNA in each group was determined by qRT-PCR. MTT colorimetry was performed to evaluate the proliferation of each group cells. Transwell assay was used to evaluate the cell invasion and migration ability. Meanwhile, the protein expression levels of SFRP1, key factors Wnt4 and β-catenin in the Wnt/β-catenin signaling pathway were determined by Western blot. Results Compared with adjacent normal tissues, miR-27a was highly expressed in colorectal cancer tissues, while SFRP1 was low expressed in colorectal cancer tissues(P<0.05). TargetScan software and dual luciferase reporter gene test showed that miR-27a targeted SFRP1. Compared with NC group, the expression of miR-27a of miR-27a mimic group increased, the proliferation, invasion and migration ability enhanced, the expression of SFRP1 protein decreased, while Wnt4 and β-catenin protein expression increased(P<0.05). Compared with miR-27a mimic group, the expression of miR-27a of miR-27a inhibitor group decreased, the proliferation, invasion and migration ability reduced, the expression of SFRP1 protein increased, while Wnt4 and β-catenin protein expression decreased(P<0.05). Conclusion miR-27a can target SFRP1, inhibit the proliferation, invasion and migration of colorectal cancer cells, mainly by up-regulating SFRP1 and blocking the downstream Wnt/β-catenin signaling pathway, which provides a new direction for clinical treatment.

abstract:
Objective To investigate the regulatory effect of artemisinin derivative dihydroartemisinin(DHA) on anti-tumor immune function of CD8+T cells induced by non-small cell lung cancer(NSCLC) cells.Methods NSCLC A549 cells were divided into DMSO control group and DHA treatment group.A549 cells were treated with DMSO and DHA at different concentrations(25,50 and 100 μmol/L),and the optimal concentration of DHA was selected to treat A549 cells for 0,24,48 and 72 h according to half maximal inhibitory concentrate(IC50).CCK-8 method and colony formation test were used to detect the effect of DHA on the proliferation and colony formation ability of A549 cells.Peripheral blood mononuclear cells(PBMCs) of healthy individuals were isolated by density gradient centrifugation.After monocytes were removed by adhesion method,A549 cells pretreated with mitomycin C were co-cultured with PBMCs at 10:1 ratio.After 2 weeks,flow cytometry was used to detect the proportion of CD8+T cells and the expression levels of perforin and granzyme B.Results Compared with the control group,the proliferation inhibition rates of A549 cells increased after treatment with 25,50 and 100 μmol/L DHA for 24 h(P<0.01).The IC50of DHA on A549 cells was 46.26 μmol/L.According to IC50concentration analysis,the inhibition rates of A549 cells treated with 50 μmol/L DHA for 0,24,48 and 72h were 1.53%,53.50%,63.84% and69.91%,and the cells inhibition rates of A548 cells increased compared with the previous observation time point,namely 0,24 and 48 h(P<0.01).The colony formation assay showed that the colony formation number of A549cells in DHA treated group decreased compared with the control group(P<0.01).Flow cytometry results showed that compared with the control group,the proportion of CD8+T cells induced by A549 cells in the co-culture system and the proportion of CD8+T cells expressing perforin and granzyme B were higher in DHA pretreatment group(P<0.01).Conclusion DHA inhibits the growth of NSCLC cells and promotes anti-tumor immune response of CD8+T cells induced by NSCLC cells.

abstract:
Objective To investigate the effect and mechanism of endothelin-1(ET-1) on atrial fibrosis in Atrial fibrillation(AF) rats. Methods Fourteen adult male SD rats were randomly divided into normal control(NC) group and Atrial fibrillation(AF) group. The rat model of Atrial fibrillation was established by injecting 0.1 ml/100g CaCl2-Ach mixture into the tail vein once a day for one week. The control group was injected with the same dose of normal saline. An electrocardiogram of normal or atrial fibrillation was recorded on the first day and the eighth day in each group, and echocardiography was used to monitor atrial size and cardiac function. The fibrosis of atrial was observed using Masson and HE staining. The expression of endothelin-1(ET-1), collagen-I(Col-I), transforming growth factor-β(TGF-β) and the store operated calcium channel(SOCC) protein Orai1, stromal interaction molecule 1(STIM1) in atrial tissue were detected by Western blot. HL-1 cells were cultured and treated with gradient concentration of ET-1 for 24 hours. Western blot was used to observe changes in the expression of TGF-β, Orai1 and STIM1 proteins in ET-1/SOCC/TGF-β signaling pathway of HL-1 cells. Small interfering RNA(siRNA) transfection method was used to knock down the expression of Orai1 in HL-1 cells, then the cells were treated with appropriate concentrations of ET-1 for 24 hours, and the expression of TGF-β protein in HL-1 cells was detected by Western blot. Results Compared with the control group, echocardiography showed a significant increase in left atrial diameter(LAD) of the heart in atrial fibrillation rats(P<0.05). The HE and Masson staining results showed significant fibrosis in the myocardial tissue of AF group rats(P<0.05), and the Western blot results indicated the expression of ET-1, Orai1, STIM1, TGF-β and COL-Ⅰ in the myocardial tissue of AF group significantly increased compared to the NC group(P<0.05). After ET-1 treatment of HL-1 cells, the protein expression of Orai1, STIM1and TGF-β increased(P<0.05), while knocking down Orai1 in HL-1 cells, ET-1 treatment no longer caused the expression of TGF-β a significant upregulation. Conclusion AF caused by atrial fibrillation results in a significant increase in ET-1 expression in atrial tissue, and ET-1/SOCC/TGF-β signal pathway promotes atrial fibrillation and fibrosis.

abstract:
Objective To investigate the molecular mechanism of ferroptosis in glioblastoma(GBM) and to provide insights for identifying new therapeutic targets. Methods GSE108474 was selected from gene expression omnibus(GEO) database and differentially expressed genes(DEGs) in GBM were obtained by using GEO2R, compared with the gene set in the Ferroptosis database(FerrDb) to identify ferroptosis-related gene. GO and KEGG enrichment analyses were conducted using DAVID database. A protein-protein interaction network was created using String website. Hub genes with high connectivity were confirmed using Cytoscape software. Prognostic and immune infiltration analyses were performed using TIMER website. RNA expression levels and gene correlation analyses were carried out using GEPIA website. Differential expression of hub gene proteins was analyzed by using the HPA database. Tumor immune characteristic correlations were examined using TISIDB database. Differences in mRNA expression of hub genes between tumor cells A172 and U251MG and normal astrocytes HA1800 were compared using the quantitative real-time PCR. Results Out of 5 331 differentially expressed genes, 114 were related to ferroptosis. GO and KEGG enrichment analysis suggested that these 114 genes might play roles in positive regulation of gene expression, and affect tumor progression through ferroptosis and autophagy pathways. 10 hub genes were identified in the protein-protein interaction network, among which cluster of differentiation 44(CD44), murine double minute 2(MDM2) and signal transducer and activator of transcription 3(STAT3) were found to be highly expressed in tumors with lower survival rates.CD44,MDM2 and STAT3mRNA expression were higher in GBM cells compared to normal cells. Protein expression of CD44, MDM2 and STAT3 was higher in high-grade glioma tissues than that in normal tissues. The expression of three genes in the tumor was negatively correlated with ferroptosis. Immune infiltration analysis revealed thatCD44,MDM2andSTAT3in the tumor were related to the infiltration of neutrophils, CD4+T cells, and dendritic cells, and the expression of three genes was related to various chemokines and their receptors. Conclusion CD44,MDM2andSTAT3may play a role in tumor ferroptosis and immune regulation, which have the potential to become a therapeutic target for GBM.

abstract:
Objective To investigate the status quo of drug resistance and influencing factors of tuberculosis in Inner Mongolia, and to provide reference for accurate prevention and control of drug-resistant tuberculosis. Methods Random sampling was used in this study. TB patients from Tuberculosis designated hospital in Inner Mongolia were included, according to the rules and drug-resistant strains were identified and tested according to relevant norms. Composition ratio or rate was calculated for statistical description, and Logistic regression model was used to analyze the influencing factors of drug resistance in TB patients. Results Among 1 321 patients, there were 936 males and 385 females, with an average age of(52.65±18.09) years. The rates of mono-resistant, multidrug-resistant(MDR), extensively drug-resistant(XDR) and total drug resistance were 19.00%, 11.58%, 11.66% and 42.24%, respectively. The highest resistance rates were observed for streptomycin(7.27%), isoniazid(4.69%), and isoniazid + streptomycin(4.47%). The drug resistance spectrum presented diversity and complexity. Compared to females, males had a higher proportion of drug resistance, and the difference was statistically significant(P<0.001). The proportion of patients who were sensitive to anti-tuberculosis drugs increased with age(P<0.05). Among different age groups, the proportion of drug-resistant patients was higher in the 20-40 age group, 40-60 age group, and 60 and above age group compared to the 0-20 age group(P<0.05). Additionally, the proportion of drug-resistant patients was higher in the 20-40 age group and 40-60 age group compared to the 60 and above age group(P<0.05). Moreover, the proportion of drug-resistant and multi-drug resistant patients was higher among patients undergoing retreatment compared to those undergoing initial treatment(P<0.001). Multivariate Logistic regression analysis showed that male gender(OR=1.48, 95%CI: 1.02-2.14), age 20-40 years(OR=2.64, 95%CI: 1.05-6.60), retreatment(OR=2.34, 95%CI: 1.70-3.22), and outpatient follow-up(OR=1.56, 95%CI: 1.05-2.33) were independent risk factors for drug-resistant tuberculosis. Conclusion Inner Mongolia has a high prevalence of MDR and overall drug-resistant tuberculosis among patients. The drug resistance profile exhibits diversity and complexity. Risk factors that contribute to drug resistance include being male, aged between 20 and 40, undergoing retreatment, and receiving outpatient follow-up. Therefore, it is necessary to further improve clinical diagnosis and treatment, promote rational use of first-line anti-tuberculosis drugs, prioritize individualized treatment, enhance health education, improve the medical insurance system, and optimize patient management approaches in order to enhance patient compliance.

abstract:
Objective To evaluate the sleep quality of patients with chronic kidney disease(CKD) and to explore the related factors of sleep disorder in patients with CKD. Methods The basic data of hospitalization patients with CKD without renal replacement therapy were prospectively collected, and the Pittsburgh sleep quality index(PSQI) scale was used to evaluate the sleep quality of patients. Patients with a PSQI score of ≤5 were divided into the normal sleep group, and patients with a PSQI score of>5 were divided into the sleep disorder group. Logistic regression analysis was used to explore the related factors of sleep disorder in patients with CKD. Results A total of 189 patients with CKD who did not receive renal replacement therapy were included, including 114 males(60.3%) and 75 females(39.7%), aged 56.5±15.23 years. The PSQI score was 7.00(5.00, 8.00), there were 58 cases in the normal sleep group and 131 cases in the sleep disorder group, and the prevalence of sleep disorder was as high as 69.3%. As the CKD stage progresses, the prevalence of sleep disorders gradually increases. There were differences between the sleep disorder group and the normal sleep group in subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disorder superposition problems, and daytime dysfunction(P<0.05), while there was no statistically significant difference in the scores of sleep medication use. Retirement or unemployed(OR=6.509, 95%CI:1.844-22.976) and women(OR=4.561, 95%CI: 1.241-16.767) were independent risk factors for sleep disorders, while eGFR(OR=0.960, 95%CI: 0.931-0.991) was a protective factor for sleep disorders,P<0.05. Conclusion The prevalence of sleep disorders in patients with chronic kidney disease without renal replacement therapy gradually increases with the decrease of eGFR and the increase of CKD stage, but they do not receive timely intervention with sleep improvement drugs. Clinicians need to focus on assessing sleep quality in women versus unemployed or retired patients with CKD.

abstract:
Objective To explore the prevalence of uremic pruritus(UP) in patients with maintenance hemodialysis(MHD) in Anhui Province and its influential factors. Methods Patients with MHD were enrolled in 27 hemodialysis centers in Anhui Province. Clinical data were compared. Results A total of 3 025 patients with MHD were included. The prevalence of UP was 63.3%, among them, mild UP 55.9% and moderate to severe UP 7.4%. The prevalence rates of UP in southern Anhui, central Anhui and northern Anhui were 75.4%,63.6% and 57.9%.The prevalence of total UP in ≤30 years, 31-50 years, 51-70 years and ≥71 years was 53.5%, 59.8%, 65.4% and 65.9%. The prevalence of total UP and moderate to severe UP increased with age(P<0.01). Age, age of dialysis, proportion of hypertension, 25(hydroxy)vitamin D3[25(OH)D3], proportion of low flux dialyzer usage and proportion of calcium-phosphorus binder usage in UP group were higher than those in the group without UP. However, the levels of diastolic blood pressure, hemoglobin(Hb) and hemodialysis filtration ratio in the UP group were lower than those in the non-UP group(P<0.05). By comparison, the age, hypertension and diabetes of patients in moderate and severe UP group were higher than those in mild UP group, while the proportion of non-calcium-phosphorus binding was lower than that in mild UP group(P<0.05). Binary Logistic regression analysis showed that high 25(OH)D3was associated with a higher risk of UP in MHD patients, and high throughput dialyzer use was associated with a lower risk of UP in MHD patients(P<0.05). Conclusion The prevalence rate of UP in maintenance hemodialysis patients in Anhui province is 63.3%. The prevalence of UP is the highest in southern Anhui, and the prevalence of total UP and moderate to severe UP increases with age. High 25(OH)D3levels are a risk factor for UP in MHD patients, and the use of high-throughput dialyzers can reduce the risk of UP in MHD patients.

abstract:
Objective To investigate the diagnostic efficacy of the nomogram model based on Chinese thyroid imaging reporting and data system(C-TIRADS) combined with shear wave elastography(SWE) and clinically independent risk factors for category IV thyroid nodules. Methods 2D-ultrasound images and SWE images of 256 patients(269 nodules) with category IV thyroid nodules were analyzed. The sensitivity, specificity, and accuracy of the diagnosis by C-TIRADS and SWE were calculated using pathological findings as the gold standard. Receiver operating characteristic(ROC) curves were plotted, and the area under the curve(AUC) was obtained. Independent risk factors for thyroid nodules were screened by univariate and multifactorial logistic regression analyses, a risk model was developed and a nomogram model was plotted, and a calibration curve analysis was used to assess the accuracy of prediction. ROC of the nomogram model was plotted, and the diagnostic efficacy of C-TIRADS, SWE and nomogram model based on independent risk factors was compared according to the AUC in category IV thyroid nodules. Results The sensitivity, specificity, and accuracy of C-TIRADS for differentiating malignant and benign nodules was 0.921, 0.724 and 0.844 respectively, the AUC was equal to 0.822 with a 95% confidence interval(95%CI) of 0.775-0.870. The sensitivity, specificity and accuracy of SWE were 0.701, 0.981, 0.814 respectively, and the AUC was 0.833(95%CI: 0.795-0.872). Multifactorial logistic regression analysis suggested that C-TIRADS classification, mean value of elasticity(E-mean) age and aspect ratio were independent risk factors for identifying benign and malignant thyroid nodules. The sensitivity, specificity and accuracy of the nomogram model established based on the above four factors were 0.957, 0.943 and 0.959, the AUC was 0.963(95%CI: 0.943-0.984), which showed a diagnostic efficacy superior to that of C-TIRADS or SWE alone. Conclusion The nomogram model, constructed based on C-TIRADS, SWE and clinically independent risk factors, can improve the efficacy in diagnosing category IV thyroid nodules, with a better clinical application value.

abstract:
Objective To evaluate the therapeutic effect of CT/MRI image fusion and usual CT guided percutaneous radiofrequency thermocoagulation of trigeminal semilunar ganglion. Methods The medical information of 88 patients diagnosed with primary trigeminal neuralgia were assembled. In accordance with different imaging guidance means, they were equally divided into the control group(trigeminal semilunar ganglion radiofrequency thermocoagulation with CT guidance) and the fusion group(trigeminal semilunar ganglion radiofrequency thermocoagulation with assistance of CT/MRI image fusion technology) at random. The puncture time, intraoperative discomfort rate, preoperative, intraoperative and postoperative visual analogue scale(VAS) score, Barrow neurological institute(BNI) pain score and postoperative complication rate were contrasted. Results The puncture operation time of the fusion group was shorter than that of the control group(P<0.05); the intraoperative and postoperative VAS and BNI scores, occurrence rate of intraoperative discomfort and postoperative complications in the fusion group were lower than those in the control group(P<0.05). Conclusion In respect of improving therapeutic effect and diminishing intraoperative discomfort and postoperative complications, CT/MRI image fusion technique is superior to CT guidance.

abstract:
Objective To explore the diagnostic value of lymphocyte subpopulations combined with chemokines in children with immunologic thrombocytopenic purpura(ITP). Methods 132 children with proposed diagnosis of ITP were collected, and the children were divided into ITP and non-ITP groups according to the diagnostic results of ITP-related clinical diagnostic criteria. 6 ml of peripheral venous blood was drawn, the levels of CD4+CD8+and CD3+were detected using flow cytometry, and the levels of chemokine(C-C motif) ligand 5(CCL5), Recombinant Chemokine(C-X-C Motif) Ligand 1(CXCL11), and monocyte chemotactic protein-1(MCP-1) were detected using enzyme-linked immunosorbent assay, the blood platelet(PLT) was measured by a fully automated cell analyzer. The children were divided into ITP and non-ITP groups according to the clinical diagnostic criteria related to ITP. The lymphocyte subpopulations and chemokine levels of the two groups of children were compared, and the correlation between lymphocyte subpopulations and chemokine levels and PLT was analyzed. The ROC method was used to evaluate the diagnostic efficacy of individual and combined detection of each indicator for ITP. Results The levels of CD4+and CD3+in the ITP group were lower than those in the non ITP group(P<0.05), while the levels of CD8+were higher than those in the non ITP group(P<0.05). The levels of CCL5, CXCL11, and MCP-1 in the ITP group were higher than those in the non ITP group(P<0.05). The correlation analysis results showed that CD4+, CD3+and platelet count were positively correlated in the ITP group(P<0.05), while CD8+, CCL5, CXCL11, MCP-1 were negatively correlated with PLT(P<0.05). The ROC analysis results showed that the cut-off values of CD4+, CD8+, CD3+, CCL5, CXCL11, and MCP-1 for the diagnosis of ITP in children were 27.13%, 24.02%, 59.88%, 41.02 ng/L, 30.18 ng/L, and 188.27 ng/L, respectively. The AUC values were 0.893, 0.880, 0.629, 0.801, 0.892, and 0.751, respectively, The AUC of the parallel diagnosis(meaning that one or more of CD4+, CD3+was below the cut-off value and/or one or more of CD8+, CCL5, CXCL11, MCP-1 was above the cut-off value at the time of parallel testing) was 0.967, indicating that one or more of them was lower than the cut off value and/or one or more of them was higher than the cut off value when tested separately. Its diagnostic efficacy was higher than that of each indicator tested separately(P<0.05). Conclusion There are significant differences in lymphocyte subpopulations and chemokines between pediatric ITP patients and non-ITP patients. CD4+, CD8+, CD3+, CCL5, CXCL11, and MCP-1 can be used for the diagnosis of pediatric ITP. Combined detection of various indicators can improve detection efficiency.

abstract:
Objective To explore the association between mammalian sterile 20-like kinase 1(MST1) gene polymorphism and haplotype and the risk of colorectal cancer, rectal cancer, and colon cancer in the Han population in Baotou area by case-control association study. Methods A total of 390 patients with colorectal cancer diagnosed by pathology and 413 normal physical examination population were collected, and 2 ml of peripheral blood was taken for subsequent gene genotyping. Single nucleotide polymorphisms(SNPs) ofMST1gene were screened according to the genetic polymorphism data of Chinese Han population provided by the National Center for Biotechnology Information-Haplotype Mapping database. Gene genotyping was performed by Taqman method. Logistic regression was used to calculate the association between each SNP and the risk of colorectal cancer, colon cancer, and rectal cancer under codominant, dominant, overdominant, and recessive genetic models. Results Four SNPs ofMST1gene were screened, namely rs8000, rs2234197, rs2267853, and rs6073629. Among them, SNP rs2234197 was associated with the risk of rectal cancer. Compared with the GG+AA genotype, the AG genotype could reduce the risk of rectal cancer, OR[95%confidence interval(CI)] =0.657(0.442-0.976). SNP rs8000 was associated with the riskof colon cancer. Compared with the TT+GT genotype, the GG genotype could reduce the risk of colon cancer [OR(95%CI)=0.425(0.182-0.992)]. Conclusion MST1gene SNP rs2234197 AG genotype and SNP rs8000 GG genotype may be protective factors for rectal cancer and colon cancer, respectively.

abstract:
Objective To construct a rat model of trigeminal neuralgia(TN) to explore the expression of high-mobility group box-1(HMGB1) in the trigeminal ganglion(TG) and the possible mechanism of HMGB1's effect on pain. Methods TN model was constructed by infraorbital nerve constriction and divided into operation group(CCI group) and Sham group, and the success of the model construction was determined through mechanical pain threshold assessment. Real time fluorescence quantitative PCR(RT-qPCR) and Western blot were used to detect high mobility group protein B1(HMGB1), Toll receptor 4(TLR4), and Nuclear Factor Kappa B(NF-κB) mRNA and protein expression in the ipsilateral trigeminal ganglion(TG) of the Sham and CCI rats. 50 mg/kg HMGB1 inhibitor glycyrrhizin(GL) was injected intraperitoneally every day for two weeks, and normal saline(NS) was used as control. The patients were divided into CCI group, CCI+NS group and CCI+GL group. HMGB1, TLR4, and NF-κB mRNA and protein expression in the ipsilateral trigeminal ganglion(TG) were detected by RT-qPCR and Western blot in CCI group, CCI+NS group, and CCI+GL group. Results The mechanical threshold on the operated side of the rat continued to decrease(P<0.05), and mechanical pain threshold identification model was successfully constructed. After chronic compressive injury to the infraorbital nerve in rats, HMGB1, TLR4, and NF-κB mRNA and protein expression in TG on the operated side increased(P<0.05); After administration of HMGB1 inhibitor Glcyrrhizin, HMGB1, TLR4, NF-κB showed a decrease(P<0.05). Conclusion HMGB1 is associated with TN, and HMGB1 may be involved in the pathogenesis of TN through TLR4/NF-κB signaling pathway.

abstract:
Objective To evaluate the effect of melatonin on nocturnal exacerbation of neuropathic pain and to explore its mechanism through the specific silencing information regulator 1(SIRT1)-brain and muscle ARNT-like protein 1(BMAL1) pathway. Methods 96 SPF-grade male C57/B6 mice were randomly divided into three groups: the sham operation(S) group, the neuropathic pain model(NP) group and the NP model + melatonin treatment(10 mg/kg)(NP+M) group; preoperative experimental mice were placed in the environment of the specified light pattern; the environment of alternating 12 h light and 12 h darkness was used for at least two weeks, and natural time was converted into the time of the award(ZT), and the starting point of the light was ZT0; only the sciatic nerve was isolated in the S group, and the mouse NP model was prepared using chronic constriction injury(CCI) of the sciatic nerve in the NP group and the NP+M group, and the NP+M group was injected with melatonin after the operation; the expression levels of SIRT1, BMAL1, and glutathione peroxidase 1(Gpx1) were detected in the spinal cord at each time point at 14 d postoperatively by Western blot. Postoperative co-staining of SIRT1 in the dorsal horn of the spinal cord with the spinal cord neuronal marker neuron-specific nuclear protein(NeuN), the microglial cell activation marker ion-calcium-binding adapter molecule 1(iba-1), and the astrocyte marker glial fibrillary acidic protein(GFAP) was carried out by immunofluorescence and iba-1 was detected at each time point to determine the activation status of microglia. Results SIRT1, BMAL1 and Gpx1 decreased in NP group mice at 14 d ZT22 postoperatively compared to ZT10 time point in NP group(P<0.05); SIRT1 and BMAL1 were elevated in NP+M group at ZT14 time point compared to ZT14 time point in NP group(P<0.05), whereas Gpx1 was elevated at ZT18 time point(P<0.05). SIRT1 was co-expressed in the dorsal horn of the spinal cord and in microglia. Compared with ZT10 time point, microglia expression decreased in NP group mice at ZT22 time point 14 d after surgery(P<0.05); compared with ZT10 time point, there was no statistically significant difference in microglia expression in NP+M group mice at ZT22 time point 14 d after surgery. Conclusion Melatonin attenuates nocturnal exacerbation of neuropathic pain by a mechanism that may be related to activation of microglia SIRT1-BMAL1 pathway protein expression.

abstract:
Objective To investigate the neurobehavioral effects of long-term mild hypothermia(MHT) combined with compound porcine cerebroside and ganglioside injection(CPCGI)after traumatic brain injury(TBI) in rats and its mechanism. Methods 36 healthy adult male SD rats were randomly divided into model group, MHT group, CPCGI group and MHT+CPCGI group. The TBI model was prepared using an electronically controlled cortical injury device. The rats in model group received an intraperitoneal injection of an equal amount of normal saline(NS, 2 ml/kg) and were treated at room temperature(37 ℃) for 48 hours. The rats in MHT group received an intraperitoneal injection of an equal amount of NS and were treated at a slightly low temperature(33.0±1.0) ℃ for 48 hours. The rats in CPCGI group received an intraperitoneal injection of an equal amount of CPCGI(0.6 ml/kg) and were treated at room temperature for 48 hours. The rats in MHT+CPCGI group received an intraperitoneal injection of an equal amount of CPCGI and were treated at a slightly low temperature for 48 hours. The sensorimotor function of rats was evaluated by modified Neurological Severity Score(mNSS). The motor and spatial memory abilities of rats were detected by Morris water maze test, and the motor function of rats was evaluated by beam walking test(BWT) and inclined-grid climbing test. The number of neurons in hippocampus was observed by Nissl staining and immunofluorescence was used to detect the expression of doublecortin(DCX) and neuronal nuclear antigen antibody(NeuN). Western blot was used to observe the protein expression of B-cell lymphoma-2(Bcl-2), Bcl-2 associated X protein(Bax) and cysteine proteinase-3(Caspase-3). Results Compared with MHT group and CPCGI group, MHT+CPCGI group had a lower mNNS score, shorter escape latency, higher times across the platform and the percentage of time in the target quadrant, higher BWT score and larger climbing angle, increased numbers of neurons, DCX and NeuN positive cells, increased Bcl-2 expression and decreased expression of Bax and Caspase-3.(P<0.05). Conclusion Long-term mild hypothermia combined with CPCGI can effectively improve the neurological deficits of TBI rats by promoting nerve regeneration and inhibiting cell apoptosis, and provide potential strategies and basis for the clinical treatment of TBI.

abstract:
Objective To investigate the expression and mechanism of long non-coding RNA(lncRNA) metastasis-associated lung adenocarcinoma transcript 1(MALAT1) and microRNA-181 a(miR-181a) in a myocardial cell oxidative stress model.Methods The expression of MALAT1 and miR-181a in peripheral blood of 30 patients with acute myocardial infarction(AMI group) and 30 healthy controls(Normal group) was detected by qRT-PCR.Pearson correlation analysis was performed to determine the correlation between MALAT1 and miR-181a in AMI.The binding sites between MALAT1 and miR-181a were predicted using the lncBase online prediction database and validated by dual-luciferase reporter assay.An oxidative stress model of myocardial cells was established by hydrogen peroxide(H2O2) treatment in AC 16 human myocardial cell line.siRNA targeting MALAT1(si-MALAT) and negative control siRNA(si-NC) were transfected into AC 16 cells,and the cells were divided into H202 treatment(H2O2) group,H2O2+si-NC group,and H2O2+si-MALAT group.Cell proliferation activity was detected by CCK-8 assay,cell apoptosis was assessed by TUNEL assay,and the expression levels of cleaved caspase-3,Bcl-2-associated X protein(Bax),and B-cell lymphoma-2(Bcl-2) were determined by Western blot.Results Compared to the Normal group,the expression of MALAT1 was upregulated and the expression of miR-181a was downregulated in the AMI group(P<0.05),and there was a negative correlation between MALAT1 and miR-181a expression.The lncBase online prediction database and dual-luciferase reporter assay results had proven that MALAT1 could target and regulate the expression of miR-181a.Compared to the H2O2group,the H2O2+si-MALAT group showed increased cell viability(P<0.05),decreased TUNEL-positive rate(P<0.05),decreased expression levels of cleaved caspase-3 and Bax(P<0.05),and increased expression level of Bcl-2(P<0.05),while the H202+si-NC group showed no significant changes(P>0.05).Conclusion LncRNA MALAT1 expression is elevated in AMI patients,which could promote oxidative stress-induced myocardial cell damage through targeted inhibition of miR-181a.

abstract:
Objective The purpose of this study was to investigate the effects of OTU deubiquitinase, ubiquitin aldehyde binding 2(OTUB2) on the activity of DEAD-box helicase 54(DDX54) and its influence on the formation of neutrophil extracellular traps(NETs) and the vitality and invasion of colorectal cancer(CRC) cells. Methods Peripheral blood neutrophils were isolated from CRC patients and healthy controls, then stimulated with phorbol-12-myristate-13-acetate(PMA) or DNase Ⅰ for 4 hours. Western blot analysis was performed to detect the expression of NETs markers, myeloperoxidase(MPO), and citrullinated histone H3(Cit-H3). The expression of OTUB2 or DDX54 was manipulated in SW480 cells, and they were co-cultured with neutrophils. The cells were divided into the following groups: Control group(without any treatment), NETs group, vector group, OTUB2 group, NETs+si-OTUB2 group(SW480 cells, SW480 cells transfected with vector, OTUB2 overexpression plasmid and si-OTUB2 were co-cultured with PMA-treated neutrophils, respectively), OTUB2+DNase Ⅰ group(SW480 cells transfected with OTUB2 overexpression plasmid co-cultured with neutrophils treated with DNase Ⅰ), si-OTUB2group, OTUB2+si-NCgroup, OTUB2+si-DDX54group(SW480 cells transfected with si-OTUB2, OTUB2 overexpressing plasmid and si-NC, OTUB2 overexpressing plasmid and si-DDX54 were co-cultured with neutrophils, respectively). ELISA was used to measure the relative expression of MPO-DNA complexes and the concentration of Cit-H3 in the supernatant of SW480 cells. MTT and Transwell assays were performed to evaluate cell viability and invasive ability. RNA sequencing was conducted to screen downstream genes regulated by OTUB2. The interaction between DDX54 and OTUB2 was validated using quantitative real-time polymerase chain reaction(qRT-PCR), co-immunoprecipitation(Co-IP), Glutathione S-Transferase(GST) pull-down, and His-tag pull-down experiments. Results Compared to healthy individuals, there was an increased formation of NETs in the peripheral blood of CRC patients. The CRC cells in the NETs group exhibited increased cell viability and invasion compared to the control group(P<0.05). In comparison to the vector group, the OTUB2 group showed increased relative expression of MPO-DNA complexes and Cit-H3, as well as increased cell viability and invasion(P<0.05). However, when compared to the OTUB2 group, the OTUB2+DNase Ⅰ group exhibited decreased relative expression of MPO-DNA complexes and Cit-H3, as well as decreased cell viability and invasion(P<0.05). Co-IP, GST pull-down, and His-tag pull-down experiments demonstrated the interaction between OTUB2 and DDX54. In comparison to the OTUB2+si-NC group, the OTUB2+si-DDX54 group showed decreased relative expression of MPO-DNA complexes and Cit-H3, as well as decreased cell viability and invasion(P<0.05). Conclusion deubiquitinating enzyme OTUB2 can increase the formation of NETs and promote CRC cell viability and invasion by up-regulating DDX54.

abstract:
Objective To investigate the effect of cysteine-rich acidic secretory protein-like protein 1(SPARC L1)on atherosclerosis(AS) plaque formation.Methods A case-control study design was used,394 patients with confirmed AS were selected as the case group,and 394 healthy medical examiners matched for age and gender were selected as the control group.The expression level of serum SPARC L1 was determined by enzyme-linked immunosorbent assay;immunohistochemistry was used to assess the expression level and localization of SPARC L1 protein in the AS plaque region,and the expression of SPARC L1 protein was also detected in the neutrophils and monocytes of peripheral blood of AS patients and normal controls;SPARCL1 overexpressing and the recombinant adenoviral vectors were constructed to inhibit SPARC L1 overexpression and expression,and the effects of SPARC L1 on cell migration were observed in the cell scratch assay using mouse macrophage cells(J774A.1) as target cells.Results Serum SPARC L1 levels in the AS patient group were lower than those in the healthy group(P<0.05);high SPARCL1 expression was detected in AS plaques and was mainly expressed in the cytoplasm of foamy cells;SPARC L1 expression levels in peripheral blood neutrophils and monocytes were lower than those in normal controls in AS patients(P<0.05);recombinant SPARC L1 overexpression and inhibition of expression of adenovirus was successfully constructed;the cell migration rate was decreased in J774A.1 cells that inhibited SPARCL1 expression and increased in J774A.1 cells that overexpressed SPARC L1(P<0.05).Conclusion SPARC L1 is highly expressed in foam cells at the site of AS lesions,which may result from compensatory recruitment of peripheral blood monocytes and neutrophils,and SPARC L1 may be involved as a protective factors for blood vessels in inhibiting the development of AS plaques.

abstract:
Objective To observe the expression and trends of tight junction proteins Occludin and zonula occluden-1(ZO-1) in blood-brain barrier(BBB) of rats with traumatic brain injury(TBI), and to explore the intervention effect of cannabidiol(CBD) on the BBB. Methods The TBI model of rat was prepared by modified “Feeney free fall method” and randomly divided into three groups: the sham-operated group(Sham group), the model group(TBI+vehicle group) and the CBD intervention group(TBI+CBD group), with 24 rats in each group. Each group was subdivided into six time points: 8 h, 1, 2, 3, 5 and 7 d after injury. The expression of Occludin and ZO-1, which are closely related to the permeability of BBB, was detected by immunohistochemistry, immunofluorescence staining and Western blot at different points. The permeability of BBB was detected by sodium fluorescein assay. Results The results of immunohistochemistry showed that compared with Sham group, the positive expression of Occludin and ZO-1 decreased with time after brain trauma(P<0.05), and both reached the lowest level at 2 d. The expression levels of Occludin and ZO-1 were up-regulated after 1 d of CBD intervention(P<0.05). Immunofluorescence staining showed a similar trend to Western blot results, with Occludin and ZO-1 fluorescence expression intensity and protein expression reduced after TBI compared with Sham group(P<0.05). And the expression levels of Occludin and ZO-1 were up-regulated after 2 d of CBD intervention(P<0.05). The results of fluorescein sodium experiment showed that the BBB integrity of brain tissue was destroyed after TBI, and the permeability increased after TBI(P<0.01). The permeability of BBB decreased after CBD intervention(P<0.05). Conclusion The expression of tight junction proteins Occludin and ZO-1 decreases after TBI, and the permeability of BBB is disrupted, and CBD intervention reverses the disruption of the BBB by TBI.

abstract:
Objective To use the GEO dataset and bioinformatics techniques, such as LASSO logistic regression, ssGSEA, and WGCNA, to screen for RA diagnostic markers and investigate the impact of earthly flavonoids in Smilax glabra Roxb. on specific immune cell infiltration, to screen for rheumatoid arthritis(RA) diagnostic markers on specific immune cell infiltration and to analyze the combination of flavonoids in Smilax glabra Roxb. and diagnostic markers. Methods The normal control group and RA gene chip were obtained from the Gene Expression Omnibus database. The R 4.3.0 WGCNA software package was used to integrate and analyze the dataset, identify co-expression modules and associated trait information, and screen key modules closely related to RA. LASSO regression analysis was performed using the glmnet package in R to identify characteristic genes for RA. The area under the receiver operating characteristic(ROC) curve was used to evaluate the diagnostic value of the characteristic genes in RA. The gene expression data of the normal control group and RA group were subjected to quantitative immune cell infiltration analysis using the GSVA, limma, and GSEABase packages in R. The chemical components of earthworm flavonoids in Smilax glabra Roxb. were analyzed based on UHPLC-Q-Exactive Orbitrap MS. The correlation between flavonoids and characteristic genes was assessed through molecular docking. Results The LASSO regression algorithm selected 5 characteristic genes(apolipoprotein D, zinc finger and BTB domain containing 16, C-C chemokine receptor type 5, matrix metalloproteinase 1, coronin-1A). The area under ROC curve of all 5 characteristic genes was greater than 0.85, which exhibited positive correlations with various immune cells. Twenty earthworm flavonoids of Smilax glabra Roxb. were identified using UHPLC-Q-Exactive/MS, and Mulberrin and Neobavaisoflavone were well combined with 5 immune characteristic genes. Conclusion Flavonoids compounds of Smilax glabra Roxb. have good combination with RA immune characteristic genes, providing a scientific basis for RA immunomodulation therapy and early diagnosis.

abstract:
Objective To explore the mechanism of ferroptosis induced by endoplasmic reticulum stress(ERs) in acute respiratory distress syndrome(ARDS).Methods In order to determine the effects of LPS on oxidative stress and Fe2+level of mouse capillary alveolar epithelial cells(MLE12 cells),the cells were treated with LPS(0,1,2,5 μg/ml) for 24 h.To verify the role of ferroptosis in lipopoly saccharide(LPS)-induced cell death,MLE12cells were divided into control(Con) group,iron removal inhibitor(Fer-1) group,LPS group and LPS+Fer-1group.LPS+Fer-1 group was pretreated with 10 μmol/L Fer-1 for 6 h,then the cells were exposed to 5 μg/ml LPS for 24 h.Con group was treated with solvent DMSO for 24 h.Fer-1 group was pretreated with 10 μmol/L Fer-1 for 6 h,and then treated with DMSO for 24 h.The cells in LPS group were exposed to 5 μg/ml LPS for 24 h.The MLE12 cells were divided into three groups:Con+Vector group,Con+sequence similarity family 134 member B(FAM134B) group,LPS+Vector group and LPS+FAM134B group.After transfected with vector or FAM134B overexpression plasmid for 48 h,the cells were exposed or not exposed to 5 μg/ml LPS for 24 h.Cell viability was measured by CCK-8.The levels of malondialdehyde(MDA),glutathione and iron,the protein levels of ferroptosis markers [cyclooxygenase 2(PTGS2),glutathione peroxidase 4(GPX4)] and ERs markers [glucose regulatory protein 78(GRP78),activated transcription factor 4(ATF4) and C/EBP homologous protein(CHOP)]were measured in different groups.In order to further confirm the results of in vitro cell experiments,40 mice were randomly divided into Con+Vector group,Con+FAM134B group,LPS+Vector group and LPS+FAM134B group,with 10 mice in each group.LPS-induced sepsis models were established in LPS+Vector group and LPS+FAM134B group,and the levels of GPX4 and ERs in lung tissue were evaluated by immunofluorescence staining and protein blot.Results LPS treatment increased the levels of PTGS2 and MDA,and decreased the levels of GPX4 and GSH in MLE12 cells in a dose-dependent manner.Compared with LPS group,the cell viability,GPX4and GSH levels in LPS+Fer-1 group increased significantly(P<0.05),while the PTGS2 protein level and MDA level decreased significantly(P<0.05).Compared with LPS+Vector group,LPS+FAM134B group significantly increased cell viability(P<0.05),decreased PTGS2 protein level(P<0.05) and increased GPX4 level(P<0.05).At the same time,the level of MDA in LPS+FAM134B group was lower than that in LPS+Vector group(P<0.05),and the level of GSH was higher than that in LPS+Vector group(P<0.05).In animal experiment,compared with LPS+Vector group,the expression levels of 4-HNE,ATF4 and CHOP in lung tissue of LPS+FAM134B group decreased significantly(P<0.05),and the expression levels of GPX4,FAM134B group increased significantly(P<0.05).Conclusion LPS induces ferroptosis and ERs in MLE12 cells in a dose-dependent manner.Activating the endoplasmic reticulum autophagy associated FAM134B receptor helps to inhibit ERs and alleviate cell ferroptosis.

abstract:
Objective To investigate the expression, synergistic relationship and clinical significance of alcohol dehydrogenase(ADH1A) and vascular endothelial growth factor-A(VEGFA) in hepatocellular carcinoma(HCC). Methods The expression and correlation of ADH1A and VEGFA in HCC and adjacent normal tissues were analyzed by GEPIA. TCGA and GSEA were used to analyze the pathway of ADH1A in HCC. The clinical and pathological data of 84 patients with HCC were collected, and 54 patients with paracancer normal tissue samples were selected as controls to analyze the correlation between ADH1A and VEGFA and clinicopathological parameters of HCC. Immunohistochemistry was used to detect the protein expression of ADH1A and VEGFA in cases and controls, and the correlation between the expression of ADH1A and VEGFA and the clinical progression and prognosis of patients with HCC was analyzed based on clinical pathological parameters and Kaplan-Meier. Results Bioinformatics analysis found that ADH1A was low-expressed in HCC and VEGFA was highly expressed in HCC, and there was a negative correlation between the two(P<0.001); immunohistochemical detection results showed that the expression of ADH1A in HCC tissue was lower than that in normal tissue adjacent to cancer(P<0.01) while the expression rate of VEGFA in HCC tissue was significantly higher than that of normal tissue adjacent to cancer(P<0.01); The recurrence rate of vascular thrombus and HCC patients in HCC group with high expression of ADH1A was lower(P<0.05). The proportion of tumor diameter>5 cm, high TNM stage, microsatellite and G2-G3 differentiation in HCC tissues in VEGFA high expression group was higher(P<0.05). Kaplan-Meier survival analysis showed that patients with high ADH1A expression and low VEGFA expression had a higher five-year survival rate. Conclusion Low expression of ADH1A and high expression of VEGFA in tumor tissues of patients with HCC indicate tumor progression and can be used as one of the prognostic evaluation indicators for patients with HCC.