ObjectiveTo explore the mechanism of Cangxi Tongbi capsules （CXTB） in regulating the p38 mitogen-activated protein kinase （p38 MAPK）/NOD-like receptor protein 3 （NLRP3）/cysteine protease-1 （Caspase-1） signaling pathway to inhibit pyroptosis of cartilage cells in knee osteoarthritis （KOA）. MethodSixty male SD rats were randomly divided into a sham operation group， a model group， low， medium， and high dose CXTB groups， and a positive control group， with 10 rats per group. The modified Hulth method was employed to establish a rat model of KOA. According to their respective assignments， rats were administered CXTB （0.25， 0.5， 1.0 g·kg^-1） and Celecoxib （24 mg·kg^-1） by gavage. The sham operation and model groups were given an equivalent volume of physiological saline. Treatment was performed once daily for 28 days. Micro-computed tomography （Micro-CT） was used to assess bone volume/total volume （BV/TV） and trabecular separation （Tb.Sp）. Joint degeneration was evaluated through hematoxylin-eosin （HE） staining， safranin-fast green （SO） staining， and Osteoarthritis Research Society International （OARSI） scoring. Western blot analysis was conducted to measure the expression levels of p38 MAPK， phosphorylated p38 MAPK （p-p38 MAPK）， NLRP3， Caspase-1， and gasdermin D （GSDMD） proteins. Real-time PCR was used to assess mRNA expression levels of p38 MAPK， NLRP3， Caspase-1， and GSDMD genes. Enzyme-linked immunosorbent assay （ELISA） was used to measure serum concentrations of inflammatory cytokines including tumor necrosis factor-alpha （TNF-α）， interleukin-1 beta （IL-1β）， and interleukin-18 （IL-18）. After knee replacement surgery， cartilage tissue was analyzed using Western blot to assess the protein expression levels of p38 MAPK， p-p38 MAPK， NLRP3， Caspase-1， and GSDMD， and Real-time PCR was used to evaluate gene expression levels of p38 MAPK， NLRP3， Caspase-1， and GSDMD. ResultMicro-CT analysis revealed significant narrowing of the joint space and increased bone spur formation in KOA rats compared with the sham operation group， with a decrease in BV/TV ratio and an increase in Tb.Sp value （P<0.01）. Serum levels of TNF-α， IL-1β， and IL-18 were elevated （P<0.01）. The protein expression levels of p-p38 MAPK， NLRP3， Caspase-1， and GSDMD in cartilage were significantly increased （P<0.01）， and the mRNA expression levels of p38 MAPK， NLRP3， Caspase-1， and GSDMD were also enhanced （P<0.01）. Significant differences in protein expression of p-p38 MAPK， NLRP3， Caspase-1， and GSDMD were observed between normal and diseased cartilage tissues after knee replacement surgery （P<0.05）， and the gene expression of p38 MAPK， NLRP3， Caspase-1， and GSDMD were also significantly different （P<0.01）. HE and SO staining showed roughened joint surfaces， reduced cartilage thickness， and disordered cellular arrangement in KOA rats. OARSI scores were significantly higher （P<0.01）. Compared with the model group， treatment with low， medium， and high concentrations of CXTB resulted in increased BV/TV ratios and decreased Tb.Sp values in the knee joints of rats （P<0.01）. HE and SO staining indicated a trend towards smoother joint surfaces and reduced OARSI scores （P<0.01）. The protein expression levels of p-p38 MAPK， NLRP3， Caspase-1， and GSDMD were notably decreased （P<0.05）， as were the mRNA expression levels of p38 MAPK， NLRP3， Caspase-1， and GSDMD （P<0.01）. Additionally， serum concentrations of TNF-α， IL-1β， and IL-18 were significantly reduced （P<0.01）. ConclusionCXTB intervention may alleviate knee joint degeneration in KOA rats and inhibit the expression of inflammatory factors and pyroptosis of cartilage cells， thereby protecting cartilage. The underlying mechanism may involve modulation of the p38 MAPK/NLRP3/Caspase-1 signaling pathway.

Pleural mesothelioma （PM） is a rare malignant tumor originating from the pleura. Most patients are already in the advanced stage at the time of diagnosis， resulting in a low overall survival rate. MicroRNA （miRNA）， as key regulators of tumor epigenetic modification， have an intertwined interactive network with PM drug resistance. The mechanisms of drug resistance in PM to chemotherapeutic drugs include increasing drug efflux， reducing drug intake， enhancing DNA repair， and altering drug targets. The mechanisms of resistance to targeted therapy drugs include activating alternative signaling pathways， establishing a favorable tumor microenvironment， and triggering epithelial-mesenchymal transition. MiRNA plays a key part in the aforementioned resistance mechanisms， with some miRNAs promoting the drug sensitivity of cancer cells， while others contribute to increased drug resistance. In light of these key regulatory functions， targeting the dysregulated expression of endogenous miRNAs in the process of resistance formation using miRNA antagonists or miRNA mimics may be an effective therapeutic strategy to reverse drug resistance in PM.

Objective:To study the trinucleotide repeats of GCN (GCA, GCT, GCC, GCG) encoding Alanine in exon 3 of the PHOX2B gene among healthy individuals from southwest China and two patients with Congenital central hypoventilation syndrome (CCHS). Methods:The number and sequence of the GCN repeats of the PHOX2B gene were analyzed by capillary electrophoresis, Sanger sequencing and cloning sequencing of 518 healthy individuals and two newborns with CCHS, respectively. Results:Among the 1036 alleles of the 518 healthy individuals, five alleles were identified, including (GCN) 7, (GCN) 13, (GCN) 14, (GCN) 15 and (GCN) 20. The frequency of the (GCN) 20 allele was the highest (94.79%). And five genotypes were identified, which included (GCN) 7/(GCN) 20, (GCN) 13/(GCN) 20, (GCN) 14/(GCN) 20, (GCN) 15/(GCN) 20, (GCN) 20/(GCN) 20. The homozygous genotypes were all (GCN) 20/(GCN) 20, and the carrier rate was 89.58%. Four GCN sequences of the (GCN) 20 homozygous genotypes were identified among the 464 healthy individuals. The GCN repeat numbers in the exon 3 of the PHOX2B gene showed no significant difference between the expected and observed values, and had fulfilled the, Hardy-Weinberg equilibrium. The genotypes of the two CCHS patients were (GCN) 20/(GCN) 25 and (GCN) 20/(GCN) 30, respectively. Conclusion:It is important to determine the GCN repeats and genotypic data of the exon 3 of the PHOX2B gene among the healthy individuals. The number of GCN repeats in 518 healthy individuals was all below 20. The selection of appropriate methods can accurately detect the polyalanine repeat mutations (PARMs) of the PHOX2B gene, which is conducive to the early diagnosis, intervention and treatment of CCHS.

Objective To create a recombinant rabies virus overexpressing IL-33 and to clarify the effect of IL-33 overexpression on the phenotypic characteristics of recombinant virus in vitro. Methods The IL-33 gene was obtained and amplified from the brain of a highly virulent strain of rabies infected mouse. It was then inserted between the G and L genes of the parental virus LBNSE genome by reversing genetic manipulation and rescuing a recombinant virus overexpressing IL-33. BSR cells or mouse NA cells were infected with recombinant rabies virus (rLBNSE-IL33) and the parental strain LBNSE. Sequencing and fluorescent antibody virus neutralization assay was employed to detect the stability of recombinant virus at multiplicity of infection=0.01. Viral titres focal forming units (FFU) were detected to plot multi-step growth curves (multiplicity of infection=0.01). Cytotoxicity assay kit was used to detect cellular activity. ELISA was adopted to identify the IL-33 in the supernatant of infected cells of different multiplicity of infection. Results Rescued rLBNSE-IL33 overexpressing IL-33 remained stable for at least 10 consecutive generations and had virus titers of approximately 10⁸ FFU/mL. rLBNSE-IL33 was able to express IL-33 at high levels in a dose-dependent manner, but no high expression of IL-33 was detected in the supernatant of cells infected by LBNSE. Examination of the titers of rLBNSE-IL33 and the parental strain LBNSE in BSR and NA cells over 5 days showed no significant differences and similar kinetic properties in growth. Overexpression of IL-33 had no significant effect on the proliferation and activity of infected cells. Conclusion Overexpression of IL-33 does not significantly affect the phenotypic characteristics of recombinant rabies virus in vitro.
Animals ; Cricetinae ; Mice ; Cell Line ; Interleukin-33/genetics* ; Rabies virus/genetics* ; Phenotype

【Objective】 To investigate the effects of 450 nm diode blue laser on the morphological changes and thermal damage of renal pelvis under different conditions. 【Methods】 An ex vivo study was conducted on a fresh porcine pelvis model (7 cm×5 cm×3 cm). The laser fiber was fixed on the mechanical arm perpendicular to the renal pelvis tissue, and the distance between them was 1-2 mm. The renal pelvis tissue was incised at a speed of 1-2 mm/s and power of 5-30 W. After the incised tissue was fixed in formalin (4%), the morphology, depth, width and coagulation thickness were observed with naked eyes and a microscope. 【Results】 The different powers had different vaporization and incision effects. When the operating distance was 2 mm, the speed was 2 mm/s and power was 5 W, the vaporization depth, width and coagulation thickness were approximately 0 9 mm, 0.25 mm and 0.35 mm, respectively. With the increase of power, the vaporization width and depth increased, and the coagulation thickness was 0.35-0.50 mm. When the power was more than 10 W, the renal pelvis tissue was easily penetrated. When the laser power was 20 W, the section of the renal pelvis showed an irregular shape of vaporization. When the operating distance was 1 mm, the whole renal pelvis tissue was easily vaporized. When it was 2 mm, a wide and safe energy treatment window was produced. 【Conclusion】 The 450 nm diode blue laser can vaporize and incise renal pelvis tissue safely and effectively, with high precision and little thermal damage. It is expected to be a new surgical tool in the treatment of renal pelvis lesions.

Objective:To analyze the difference of clinical characteristics and outcomes of infants with moderate and severe pediatric acute respiratory distress syndrome(PARDS)diagnosed according to baseline oxygenation index(OI) in pediatric intensive care unit(PICU).Methods:Second analysis of the data collected from the "Efficacy of pulmonary surfactant (PS) in the treatment of children with moderate and severe ARDS" program.Retrospectively compare of the differences in clinical data such as general condition, underlying diseases, OI, mechanical ventilation, PS administration and outcomes among infants with moderate and severe PARDS divided by baseline OI who admitted to PICUs at 14 participating tertiary hospitals from 2016 to December 2021.Results:Among the 101 cases, 55 cases (54.5%) were moderate and 46 cases (45.5%) were severe PARDS.The proportion of male in the severe group (50.0% vs.72.7%, P=0.019) and the pediatric critical illness score(PCIS)[72 (68, 78) vs.76 (70, 80), P=0.019] were significantly lower than those in the moderate group, while there was no significant difference regarding age, body weight, etiology of PARDS and underlying diseases.The utilization rate of high-frequency ventilator in the severe group was significantly higher than that in the moderate group (34.8% vs.10.9%, P=0.004), but there was no significant difference in PS use, fluid load and pulmonary complications.The 24 h OI improvement (0.26±0.33 vs.0.04±0.34, P=0.001) and the 72 h OI improvement[0.34 (-0.04, 0.62) vs.0.15 (-0.14, 0.42), P=0.029)]in the severe group were significantly better than those in the moderate group, but there was no significant difference regarding mortality, length of hospital stay and intubation duration after diagnosis of PARDS between the two groups. Conclusion:In moderate and severe(divided by baseline OI) PARDS infants with invasive mechanical ventilation, children in severe group have better oxygenation improvement in the early stage after PARDS identified and are more likely to receive high frequency ventilation compared to those in moderate group.Baseline OI can not sensitively distinguish the outcomes and is not an ideal index for PARDS grading of this kind of patient.

Objective:To analyze the risk factors for heart failure in patients with hemodialysis, and to construct a nomogram model.Methods:The clinical data of 218 patients with hemodialysis in Xianyang Central Hospital from January 2021 to April 2022 were retrospectively analyzed. Among them, 83 cases developed heart failure (heart failure group), and 135 cases did not develop heart failure (control group). The relevant clinical data were recorded, including age, sex, body mass index, disease duration, concurrent infection, blood calcium, blood phosphorus, soluble CD 146 (sCD 146), soluble growth-stimulated expression gene 2 protein (sST2), N-terminal brain natriuretic peptide precursor (NT-proBNP), time-averaged urea concentration (TACurea), tumor necrosis factor α (TNF-α), blood creatinine and 24 h urine volume. Receiver operating characteristic (ROC) curve was used to analyze the efficacy of each index in predicting heart failure in patients with hemodialysis. Multivariate Logistic regression was used to analyze the independent risk factors of heart failure in patients with hemodialysis. R language software 4.0 "rms" package was used to construct the nomogram model for predicting the heart failure in patients with hemodialysis, the calibration curve was internally validated, and the decision curve was used to evaluate the predictive efficacy of the nomogram model. Results:There were no statistical difference in gender composition, age, body mass index, disease duration, 24 h urine volume and blood creatinine between the two groups ( P>0.05); the rate of concurrent infection, blood phosphorus, sCD 146, sST2, NT-proBNP, TNF-α and TACurea in heart failure group were significantly higher than those in control group: 39.76% (33/83) vs. 8.89% (12/135), (1.53 ± 0.34) mmol/L vs. (1.27 ± 0.24) mmol/L, (43.60 ± 10.24) μmol/L vs. (28.08 ± 7.99) μmol/L, (49.00 ± 9.41) μg/L vs. (34.53 ± 8.05) μg/L, (38.57 ± 6.79) μg/L vs. (29.72 ± 5.64) μg/L, (5.18 ± 0.92) μg/L vs. (4.07 ± 1.13) μg/L and (24.28 ± 4.37) mmol/L vs. (17.96 ± 2.52) mmol/L, the blood calcium was significantly lower than that in control group: (1.95 ± 0.36) mmol/L vs. (2.31 ± 0.39) mmol/L, and there were statistical differences ( P<0.01). ROC curve analysis result showed that the optimal cut-off values of blood calcium, blood phosphorus, sCD 146, sST2, NT-proBNP, TNF-α and TACurea for heart failure in patients with hemodialysis were 2.01 mmol/L, 1.42 mmol/L, 34.15 μmol/L, 40.37 μg/L, 35.37 μg/L, 4.33 μg/L and 20.74 mmol/L. Multivariate Logistic regression analysis result showed that the blood calcium (≤2.01 mmol/L), blood phosphorus (>1.42 mmol/L), sCD 146 (>34.15 μmol/L), sST2 (>40.37 μg/L), NT-proBNP (>35.37 μg/L), TNF-α (>4.33 μg/L) and TACurea (>20.74 mmol/L) were independent risk factors for heart failure in patients with hemodialysis ( OR = 1.183, 1.582, 1.915, 1.105, 1.459, 1.347 and 1.717; 95% CI 1.102 to 1.191, 1.274 to 1.868, 1.716 to 2.105, 1.072 to 1.141, 1.225 to 1.703, 1.132 to 1.574 and 1.482 to 1.935; P<0.05 or <0.01). The blood calcium, blood phosphorus, sCD 146, sST2, NT-proBNP, TNF-α and TACurea were used as predictors to construct a nomogram model for predicting heart failure in patients with hemodialysis. Internal validation result showed that the nomogram model predicted the heart failure with good concordance in patients with hemodialysis (C-index = 0.811, 95% CI 0.675 to 0.948); the nomogram model predicted the heart failure in patients with hemodialysis at a threshold>0.18, provided a net clinical benefit, and all had higher clinical net benefits than blood calcium, blood phosphorus, sCD 146, sST2, NT-proBNP, TNF-α and TACurea. Conclusions:The nomogram model constructed based on blood calcium, blood phosphorus, sCD 146, sST2, NT-proBNP, TNF-α and TACurea has better clinical value in predicting the heart failure in patients with hemodialysis.

Objective:To investigate the radiation dosimetry and biodistribution of 68Ga-FAPI-04 PET/CT in patients with hepatobiliary tumor. Methods:A total of six patients with hepatic lesions who underwent PET/CT examination in Peking Union Medical College Hospital were enrolled. After intravenous injection of radiotracer 68Ga-FAPI-04 at (170.57 ± 14.43) MBq, whole-body imaging were performed at the time points of 3, 10, 15, 20, 30 and 60 min, respectively. Biodistribution pattern was observed. Regions of interest were manually delineated. Radiation dosimetry of all target organs were calculated by Olinda/EXM software. Results:The radioactive uptake dissipated gradually in liver whereas it was relatively stable in tumor lesions. The average SUV max of tumor lesions reached the maximum value (13.87± 2.55) at 20 min after injection. The target-to-background ratio increased with time, reaching the maximum value (10.09 ± 8.17) at 30 min after injection. The average effective dose in total body was (0.020 ± 0.002) mSv/MBq and organ with the highest effective dose was bladder wall at (0.146 ± 0.035) mSv/MBq. Conclusions:The effective dose in total body of 68Ga-FAPI-04 was similar to that of 18F-FDG. 68Ga-FAPI-04 is expected to be a PET/CT radiotracer for hepatobiliary tumors in consideration of rapid tumor uptake, low accumulation of liver background, and no influence of blood sugar levels.

Objective:To analyze and evaluate the effect of OX40L as a potential adjuvant for H7N9 whole-virion inactivated vaccine (WIV).Methods:Fifty BALB/c mice were randomly divided into five groups and immunized intramuscularly with PBS (control group) and 1.5 μg WIV alone or in combination with 0.6, 1.8 or 3.0 μg Fc-fused OX40L (OX40L/Fc) adjuvant. Three weeks after immunization, IgG, IgG1 and IgG2 titers were measured by ELISA and hemagglutination inhibition (HI) assay. Moreover, the mice were challenged with 50×median lethal dose (LD 50) of homologous virus and the changes in mouse body weight and survival rate were recorded to evaluate the effects of OX40L. Flow cytometry was used to analyze the mechanism of OX40L as an adjuvant 7 d after immunization. Results:Compared with immunization with WIV alone, co-immunization of WIV with OX40L/Fc induced higher antigen-specific IgG in mice. The geometric mean titers (lgGMT) of antibodies induced by 0.6, 1.8 and 3.0 μg OX40L/Fc reached 3.79, 4.40 and 4.20, respectively. WIV combined with OX40L/Fc induced high levels of IgG1 and IgG2a without influencing Th1/Th2 balance. HI antibodies were also higher in WIV+ 1.8 μg OX40L/Fc and WIV+ 3.0 μg OX40L/Fc groups than in WIV group (6.25±0.50 and 5.70±0.97 vs 3.00±0.97, both P<0.05). WIV combined with 1.8 or 3.0 μg OX40L/Fc could protect 80% or 75% of mice against lethal challenge with H7N9 and result in less weight loss as compared with WIV alone. The most effective dose of OX40L/Fc was 1.8 μg. Flow cytometry showed that WIV (0.6, 1.8, 3.0 μg) in combination with OX40L/Fc enhanced the proliferation of T follicular helper cells (Tfh) through promoting the expression of CXCR5 and PD-1 as compared with WIV alone (all P<0.05). Conclusions:This study suggested that OX40L was beneficial to potent antibody responses induced by H7N9 WIV through promoting Tfh cell proliferation.

Objective:To prepare 68Ga-fibroblast activation protein inhibitor (FAPI)-04, and evaluate its biodistribution and imaging characteristics in animals and healthy volunteers, in order to investigate the clinical translation potential. Methods:68Ga-FAPI-04 was synthesized by a manual method and its radiolabeling yield, radiochemical purity, and stability ( in vivo and in vitro) were analyzed. ICR mice ( n=16) were scarified at 5, 30, 60 and 120 min postinjection of 68Ga-FAPI-04 (1.11 MBq) to measure radioactive counts in main organs. The dynamic mircoPET imaging was acquired for 60 min on 3 ICR mice, and tumor imaging capabilities were examined with nude mice bearing HepG2 tumors. Furthermore, 2 healthy volunteers (1 male with age of 64 years, 1 female with age of 56 years) were recruited for the investigation of probe biodistribution in humans. A serial whole-body dynamic PET/CT scan was performed immediately following injection. Results:68Ga-FAPI-04 was synthesized within 20 min with the radiochemical yield of (68.7±4.0)% (decay corrected). The radiochemical purities of 68Ga-FAPI-04 were over 99% and the products were stable for 180 min in vitro and for 90 min in blood. 68Ga-FAPI-04 was mainly cleared through urinary tracts, while other organs only showed mild tracer accumulation. MicroPET imaging showed high uptake of 68Ga-FAPI-04 in the tumor tissue of mice, and the ratio of tumor/liver was 2.14±0.01 (35 min). The PET/CT imaging results of healthy volunteers revealed 68Ga-FAPI-04 could be quickly cleared. Conclusion:68Ga-FAPI-04 has many advantages for PET imaging, such as easy labeling, good stability, quick clearance and low background signals in the liver, which can be used as an attractive PET tracer for detection hepatocellular carcinoma.