BACKGROUND:In the repair of large bone defects,a variety of factors such as seed cell passaging can cause senescence of osteoblasts,leading to a reduction in osteogenic differentiation activity after implantation of tissue-engineered bone.In recent years,a novel mechanism involving primary cilia in cell senescence has been widely studied,but the primary cilia-related mechanism of"passage senescence-reduced osteogenic activity"is not fully understood.OBJECTIVE:To explore the possible mechanisms by which primary cilia regulate the senescence of MC3T3-E1 cells.METHODS:The osteoblast precursor cell lines MC3T3-E1 were passaged to 10th generation cells(early passage)and 40th generation cells(late passage).siRNA was used to silence IFT88 to inhibit primary cilia formation.The cells were than grouped into passage 10 group,passage 40 group,passage 10+siRNA IFT88 group,and passage 40+siRNA IFT88 group.RT-PCR and western blot assays were used to detect the expression of the aging marker P16(CDKN2A),the osteogenic activity markers bone morphogenetic protein 2 and alkaline phosphatase,and the Hedgehog pathway IHH expression.Alizarin red staining and primary cilia immunofluorescence staining were performed.Spearman correlation analysis was conducted to analyze primary cilia positive rate and IHH and bone morphogenetic protein 2 expression.RESULTS AND CONCLUSION:(1)The expression of CDKN2A(P16)in the passage 10 group was significantly higher than in the passage 40 group,but the difference disappeared after siRNA IFT88 intervention.(2)Meanwhile,the positive rate of primary cilia cells in the passage 10 group were higher than in the passage 40 group,while siRNA IFT88-significantly inhibited the expression of primary cilia in both passage 10 and passage 40 cells.(3)The transcriptional activity and protein expression of bone morphogenetic protein 2 and alkaline phosphatase in the passage 10 group were higher than those in the passage 40 group.After inhibiting the expression of primary cilia with siRNA,the above differences were reduced or disappeared.(4)The positive rate of primary cilia cells was correlated with IHH and bone morphogenetic protein 2 protein expression.To conclude,primary cilia mediate the replicative senescence of osteogenic MC3T3-E1 cells and regulate osteogenic differentiation ability.

Objective To identify novel biomarkers for predicting the risk of latent tuberculosis infection(LTBI)activation using bio-informatics and machine-learning algorithms and to establish a risk model.Methods The GSE112104 and GSE193777 datasets were obtained from the Gene Expression Omnibus.Differential gene expression and weighted gene co-expression network analyses were per-formed to identify ferroptosis-related differentially expressed genes(FRG-DEGs)associated with LTBI activation.Three machine-learning algorithms,least absolute shrinkage and selection operator,support vector machine-recursive feature elimination,and random forest,were used to identify ferroptosis-related hub genes(FRG-hubs).The reliability of these genes was validated using independent validation datasets and reverse transcription polymerase chain reaction(PCR).A risk model was established using R software.Results In the GSE 112104 dataset,296 genes were upregulated and 1 569 genes were downregulated in active tuberculosis compared to those in LTBI.Among the LTBI progressors,506 genes were upregulated and 1 132 genes were downregulated.Weighted correlation network analysis identified five gene modules,with the blue module showing the strongest correlation with LTBI activation(cor=0.62,P=0.000 04),con-taining 1 340 genes.Intersections with 728 ferroptosis-related genes resulted in eight FRG-DEGs.The machine-learning algorithms iden-tified four FRG-hubs:PLA2G6,GLS2,JUN,and AMN,whose expression decreased with LTBI activation.Reverse transcription PCR con-firmed this trend.A risk model based on these genes yielded an area under the curve of 0.98 to 1.00.Conclusion This study successfully identified novel biomarkers for predicting the risk of LTBI activation and developed an accurate predictive risk model.

BACKGROUND:In the repair of large bone defects,a variety of factors such as seed cell passaging can cause senescence of osteoblasts,leading to a reduction in osteogenic differentiation activity after implantation of tissue-engineered bone.In recent years,a novel mechanism involving primary cilia in cell senescence has been widely studied,but the primary cilia-related mechanism of"passage senescence-reduced osteogenic activity"is not fully understood.OBJECTIVE:To explore the possible mechanisms by which primary cilia regulate the senescence of MC3T3-E1 cells.METHODS:The osteoblast precursor cell lines MC3T3-E1 were passaged to 10th generation cells(early passage)and 40th generation cells(late passage).siRNA was used to silence IFT88 to inhibit primary cilia formation.The cells were than grouped into passage 10 group,passage 40 group,passage 10+siRNA IFT88 group,and passage 40+siRNA IFT88 group.RT-PCR and western blot assays were used to detect the expression of the aging marker P16(CDKN2A),the osteogenic activity markers bone morphogenetic protein 2 and alkaline phosphatase,and the Hedgehog pathway IHH expression.Alizarin red staining and primary cilia immunofluorescence staining were performed.Spearman correlation analysis was conducted to analyze primary cilia positive rate and IHH and bone morphogenetic protein 2 expression.RESULTS AND CONCLUSION:(1)The expression of CDKN2A(P16)in the passage 10 group was significantly higher than in the passage 40 group,but the difference disappeared after siRNA IFT88 intervention.(2)Meanwhile,the positive rate of primary cilia cells in the passage 10 group were higher than in the passage 40 group,while siRNA IFT88-significantly inhibited the expression of primary cilia in both passage 10 and passage 40 cells.(3)The transcriptional activity and protein expression of bone morphogenetic protein 2 and alkaline phosphatase in the passage 10 group were higher than those in the passage 40 group.After inhibiting the expression of primary cilia with siRNA,the above differences were reduced or disappeared.(4)The positive rate of primary cilia cells was correlated with IHH and bone morphogenetic protein 2 protein expression.To conclude,primary cilia mediate the replicative senescence of osteogenic MC3T3-E1 cells and regulate osteogenic differentiation ability.

Objective To identify novel biomarkers for predicting the risk of latent tuberculosis infection(LTBI)activation using bio-informatics and machine-learning algorithms and to establish a risk model.Methods The GSE112104 and GSE193777 datasets were obtained from the Gene Expression Omnibus.Differential gene expression and weighted gene co-expression network analyses were per-formed to identify ferroptosis-related differentially expressed genes(FRG-DEGs)associated with LTBI activation.Three machine-learning algorithms,least absolute shrinkage and selection operator,support vector machine-recursive feature elimination,and random forest,were used to identify ferroptosis-related hub genes(FRG-hubs).The reliability of these genes was validated using independent validation datasets and reverse transcription polymerase chain reaction(PCR).A risk model was established using R software.Results In the GSE 112104 dataset,296 genes were upregulated and 1 569 genes were downregulated in active tuberculosis compared to those in LTBI.Among the LTBI progressors,506 genes were upregulated and 1 132 genes were downregulated.Weighted correlation network analysis identified five gene modules,with the blue module showing the strongest correlation with LTBI activation(cor=0.62,P=0.000 04),con-taining 1 340 genes.Intersections with 728 ferroptosis-related genes resulted in eight FRG-DEGs.The machine-learning algorithms iden-tified four FRG-hubs:PLA2G6,GLS2,JUN,and AMN,whose expression decreased with LTBI activation.Reverse transcription PCR con-firmed this trend.A risk model based on these genes yielded an area under the curve of 0.98 to 1.00.Conclusion This study successfully identified novel biomarkers for predicting the risk of LTBI activation and developed an accurate predictive risk model.

Cancer stands as one of the predominant causes of mortality globally, necessitating ongoing efforts to develop innovative therapeutics. Historically, natural products have been foundational in the quest for anticancer agents. Bulbocodin D (BD) and Bulbocodin C (BC), two bibenzyls derived from Pleione bulbocodioides (Franch.) Rolfe, have demonstrated notable in vitro anticancer activity. In human lung cancer A549 cells, the IC_50s for BD and BC were 11.63 and 11.71 μmol·L^-1, respectively. BD triggered apoptosis, as evidenced by an upsurge in Annexin V-positive cells and elevated protein expression of cleaved-PARP in cancer cells. Furthermore, BD and BC markedly inhibited the migratory and invasive potentials of A549 cells. The altered genes identified through RNA-sequencing analysis were integrated into the CMap dataset, suggesting BD's role as a potential signal transducer and activator of transcription 3 (STAT3) inhibitor. SwissDock and MOE analyses further revealed that both BD and BC exhibited a commendable binding affinity with STAT3. Additionally, a surface plasmon resonance assay confirmed the direct binding affinity between these compounds and STAT3. Notably, treatment with either BD or BC led to a significant reduction in p-STAT3 (Tyr 705) protein levels, regardless of interleukin-6 stimulation in A549 cells. In addition, the extracellular signal-regulated kinase (ERK) was activated after BD or BC treatment. An enhancement in cancer cell mortality was observed upon combined treatment of BD and U0126, the MEK1/2 inhibitor. In conclusion, BD and BC emerge as promising novel STAT3 inhibitors with potential implications in cancer therapy.
Humans ; Lung Neoplasms/metabolism* ; STAT3 Transcription Factor/metabolism* ; Antineoplastic Agents/chemistry* ; A549 Cells ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation

8-hydroxy-2'-deoxyguanosine (8-OHdG) is a sensitive and stable biomarker for evaluating DNA oxidative damage. A rapid and sensitive colloidal gold immunochromatographic strip was developed for 8-OHdG detection by a competitive method. The sample pad (glass cellulose film), bonding pad (glass cellulose film), nitrocellulose film and absorbent pad were pasted on the polyvinyl chloride (PVC) base plate to construct the test strip. Colloidal gold (AuNPs) was prepared by the reduction of chloroauric acid with sodium citrate. 8-OHdG antibody (Ab) was coated on the outer layer of AuNPs to form Ab@AuNPs as a probe. Bovine serum albumin (BSA) and 8-OHdG were conjugated with carbodiimide hydrochloride to prepare an artificial antigen, which was used as the coating antigen of detection line. Goat anti mouse polyclonal antibody IgG was used as the coating antibody of control line. The experimental parameters were optimized including the type of nitrocellulose membrane, the formula of loading solution, and the spraying amount of gold labeled antibody. The results showed that the appropriate nitrocellulose membrane was CN 95. The optimal loading solution included BSA (1%), Tween-20 (3%), sucrose (3%) and NaCl (0.9%). The optimal spraying amount of gold labeled antibody was 4 μL. 8-OHdG can be detected by the strip under visible light, and the level of 8-OHdG in urine can be preliminarily determined by comparing the color intensity of T line and C line. The 8-OHdG concentration in urine was further calculated by the gray value of T line and the threshold of detection was 2.55 μg/L. This colloidal gold immunochromatographic strip is simple, rapid and specific for detecting 8-OHdG in human urine to preliminarily evaluate the human status.
8-Hydroxy-2'-Deoxyguanosine ; Animals ; Antibodies, Monoclonal ; Gold ; Gold Colloid/chemistry* ; Metal Nanoparticles ; Mice ; Sensitivity and Specificity

Objective To explore the value of various diffusion parameters obtained from monoexponential, biexponential and stretched exponential diffusion-weighted imaging models in assessing hepatic fibrosis in chronic hepatitis B(CHB). Methods A total of 52 patients who were diagnosed hepatitis B by the markers of hepatitis and were confirmed by liver biopsy pathology were prospectively recruited between June 2014 and May 2016 in People's Hospital of Henan Province. Concomitantly, 30 healthy volunteers who had no history of hepatitis B and liver dysfunction were enrolled in the control group. All patients underwent multi-b values DWI on a 3.0 T MRI unit. ADC was calculated by using the monoexponential model. True diffusion coefficient(Dt),pseudo diffusion coefficient(Dp)and fraction of perfusion(f)were calculated by using the biexponential model.Distributed diffusion coefficient(DDC)and water molecular diffusion heterogeneity index(α)were calculated by using the stretched exponential model. Liver biopsy specimens were staged according to the degree of hepatic fibrosis (S0 to S4). The Kruskal-Wallis test was employed for the comparison of each parameter among the control group and the fibrosis stage groups. The Mann-Whitney U test was adopted to compare each parameter between fibrosis stage≤S1and≥S2,between≤S2 and≥S3.Spearman rank correlation coefficients were obtained to assess the correlation of the parameters with the fibrosis stages.ROC analysis was used to evaluate the performance of various parameters in predicting stage≥S2 and≥S3.Results The hepatic fibrosis stage distributions were as follows:1 cases with S0,9 cases with S1,22 cases with S2,11 cases with S3,9 cases with S4.ADC,Dt,f and DDC values all showed significant difference among the control group and groups S1,S2,S3,S4(all P<0.05), while Dp and α values showed no significant difference(P>0.05). Dt, DDC and ADC showed a moderate negative correlation with the fibrosis stage(r=-0.630,-0.603 and-0.464,respectively,all P<0.01),and f showed a mild negative correlation with the fibrosis stage(r=-0.379,P<0.05),while Dp and α values showed no correlation with the fibrosis stages(all P>0.05).The ADC, Dt, f and DDC values all showed significant difference between stage≥S2 and≤S1,between stage≥S3 and≤S2(all P<0.05),and the parameter values of the former were greater than those of the latter. While Dp and α values showed no significant difference among both groups(all P>0.05).The AUCs of ADC,Dt,f and DDC values for detecting fibrosis stage ≥S2 were 0.738,0.835,0.740 and 0.831, and the AUCs of ADC, Dt, f and DDC values for detecting fibrosis stage≥S3 were 0.716, 0.811, 0.672 and 0.798. Conclusion The Dt derived from biexponential and DDC derived from stretched exponential DWI could be useful for the staging of hepatic fibrosis in CHB.

Objective To explore the correlation between changes of striatal dopamine D2receptors non-displaceable binding potential (BPND) in 11C-Raclopride PET-CT and brain regional homogeneity (ReHo) derived from resting state functional MRI (fMRI) in patients with first-episode major depressive disorder (MDD) . Methods Patients with first-episode MDD and age and sex matched healthy volunteers accepted brain 11C-Raclopride PET-CT and resting state fMRI. MIAKAT based on MATLAB and Rest 1.8 were used to calculate BPNDof brain dopamine D2receptors and brain ReHo, respectively. Changes of striatal dopamine D2receptors BPNDand brain ReHo values were analyzed by paired-sample t test and two independent sample t-test, and Pearson correlation analysis was used to analyze the correlation between BPNDand ReHo. Results Twenty patients with first-episode MDD were enrolled as MDD group, and 20 healthy volunteers as the control group. The two groups were all right-handed, and there were no statistical differences for age (t =1.33,P=0.19)and gender(χ2=0.10,P=0.75). Compared with the control group, the ReHo in MDD patients increased in the bilateral striatum (caudate nucleus and putamen), bilateral medial prefrontal lobes, and right thalamic (27 to 56 voxels, P＜0.05) and decreased in the left middle frontal gyrus, the left anterior cingulate, the left hippocampal and the right amygdala (21 to 35 voxels, P＜0.05). In addition, compared with the control group, BPNDof bilateral caudate nucleus and putamen dopamine D2receptors in the MDD group were decreased (t=－4.41 to －3.13, P＜0.05). Furthermore, there was a negative correlation between D2 receptors BPNDand ReHo of bilateral caudate nucleus and putamen (r=－0.81 to－0.62, P＜0.05). And there was a negative correlation between ReHo of the bilateral medial prefrontal lobes and BPNDof the same lateral caudate nucleus and putamen D2receptors in the MDD group (r=－0.86 to－0.52, P＜0.05). Besides, ReHo of the left middle frontal gyrus, right thalamic, left anterior cingulate, left hippocampal and right amygdala had no correlation with the D2receptors BPNDof the striatum in the MDD group (－0.27 to 0.39, P＞0.05). Conclusion There were abnormal levels of dopamine neurotransmitter in the cerebral striatum regions and abnormal brain activities in the brain region associated with dopamine reward circuit in the first-episode MDD patients, and there was a correlation between the two abnormalities.

OBJECTIVETo observe the difference in acupuncture for pain threshold at different time points among the groups of 9 TCM constitutions.

METHODSThe cross-sectional survey was adopted to investigate TCM constitutions among 600 subjects and determine 9 TCM constitution types (neutral constitution, qi-deficiency constitution, yang-deficiency constitution, yin-deficiency constitution, phlegm-damp constitution, damp-heat constitution, blood-stagnation constitution, qi-stagnation constitution, special diathesis constitution). The same acupuncture manipulation was applied to Zusanli (ST 36) on the left side in the subjects and the needle was retained for 30 min. The tenderness threshold was detected with 2390 type Von Frey apparatus at different time points, named before acupuncture, at the moment after qi arrival, in 10 min of needle retaining, in 30 min of needle retaining and in 15 min after needle withdrawal in the subjects of 9 TCM constitutions.

RESULTSThe interactive effect happened between the constitution type and time point (P < 0.05). Among the groups of 9 TCM constitutions, the pain threshold values at the moment after qi arrival (except blood-stagnation constitution, qi-stagnation constitution, special diathesis constitution) in 10 min of needle retaining and in 30 min of needle retaining were increased as compared with those before acupuncture separately (P < 0.01), among which, the value increase was the most significant in 30 min of needle retaining. The differences in the pain thresholds were significant in 15 min after needle withdrawal in the groups of neutral constitution and damp-heat constitution as compared with those before acupuncture (both P < 0.01). In 10 min of needle retaining and in 30 min of needle retaining, as compared with the group of neutral constitution, the changes in pain thresholds of the rest abnormal constitutions were apparently lower (all P < 0.05).

CONCLUSIONAcupuncture at Zusanli (ST 36) presents different effects among the groups of different constitution types. The effect maintaining durations are different.

Acupuncture Points ; Acupuncture Therapy ; Adolescent ; Adult ; Cross-Sectional Studies ; Female ; Humans ; Male ; Pain Management ; Pain Threshold ; Yang Deficiency ; therapy ; Yin Deficiency ; therapy ; Young Adult

BACKGROUNDThe diagnosis of liver fibrosis is a difficult task at any time using conventional clinical imaging. Intravoxel incoherent motion (IVIM) can be used to investigate both diffusion and perfusion changes in tissues. This study was designed to determine the value of IVIM in the diagnosis and staging of liver fibrosis.

METHODSIVIM examinations were performed on a GE 3.0T MR scanner in 25 patients with liver fibrosis and 25 healthy volunteers as the control group. Patients with liver fibrosis diagnosis were confirmed by pathology and staged on a scale of F0-4. The standard ADC values and the values of a biexponential model (slow ADC (Dslow), fast ADC (Dfast) and fraction of fast ADC (FF)) were measured in three liver regions per person. The mean standard ADC values, Dslow values, Dfast values and FF values from the study group were compared among the right posterior hepatic lobe, right anterior hepatic lobe and medial segment of the left lobe. Receiver Operating Characteristic (ROC) curves and independent-samples t-tests were used to calculate the mean standard ADC values, Dslow values, Dfast values and FF values from the study group and the control group. Spearman rho correlation analysis was used for the stage of liver fibrosis. The liver fibrosis stages between the groups F0-1 and F2-4, the groups F0-2 and F3-4 were compared.

RESULTSAmong the liver fibrosis, there was no significant difference in the mean standard ADC values, Dslow values, Dfast values, and FF values obtained from the right posterior hepatic lobe, right anterior hepatic lobe and medial segment of the left lobe. Using ROC analysis, the Area Under the Curve (AUC) values of standard ADC, Dslow, Dfast, FF were all between 0.7 to 0.9. The mean standard ADC values, Dslow values, Dfast values and FF values of the liver in the study group were significantly lower than the values in the control group (P < 0.05). As the stage of the fibrosis increased, the values decreased by Spearman rho correlation analysis. The mean values (standard ADC, Dslow, Dfast, and FF) of liver fibrosis stages between the groups F0-1 and F2-4, the groups F0-2 and F3-4 showed significant differences (P < 0.05).

CONCLUSIONSIVIM can reflect the conditions of perfusion and diffusion in liver fibrosis and thus distinguish between normal liver and liver fibrosis. The IVIM technique may serve as a valuable tool for detecting and characterizing liver fibrosis, and monitoring its progression in a noninvasive manner.

Adult ; Aged ; Diffusion Magnetic Resonance Imaging ; methods ; Female ; Humans ; Liver ; pathology ; Liver Cirrhosis ; diagnosis ; Male ; Middle Aged