This study aims to integrate data mining techniques of single cell transcriptomics and spatial transcriptomics, along with animal experiment validation, so as to systematically characterize the protective effects of Jianpi Tongluo Formula(JTF) on the cartilage in knee osteoarthritis(KOA) and elucidate the underlying molecular mechanisms. Single cell transcriptomics and spatial transcriptomics datasets(GSE254844 and GSE255460) of the cartilage tissue obtained from KOA patients were analyzed to map the single cell-spatial heterogeneity and identify key pathogenic factors. After that, a KOA rat model was established via knee joint injection of papain. The intervention effects of JTF on the expression features of these key factors were assessed through real-time quantitative polymerase chain reaction(PCR), Western blot, and immunohistochemical staining. As a result, the integrated single cell and spatial transcriptomics data identified distinct cell subsets with different pathological changes in different regions of the inflamed cartilage tissue in KOA, and their differentiation trajectories were closely related to the inflammatory fibrosis-like pathological changes of chondrocytes. Accordingly, the expression levels of the two key effect targets, namely nuclear receptor coactivator 4(NCOA4) and high mobility group box 1(HMGB1) were significantly reduced in the articular surface and superficial zone of the inflamed joints when JTF effectively alleviated various pathological changes in KOA rats, thus reversing the abnormal chondrocyte autophagy level, relieving the inflammatory responses and fibrosis-like pathological changes, and promoting the repair of chondrocyte function. Collectively, this study revealed the heterogeneous characteristics and dynamic changes of inflamed cartilage tissue in different regions and different cell subsets in KOA patients. It is worth noting that NCOA4 and HMGB1 were crucial in regulating chondrocyte autophagy and inflammatory reaction, while JTF could reverse the regulation of NCOA4 and HMGB1 and correct the abnormal molecular signal axis in the target cells of the inflamed joints. The research can provide a new research idea and scientific basis for developing a personalized therapeutic schedule targeting the spatiotemporal heterogeneity characteristics of KOA.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Osteoarthritis, Knee/pathology* ; Humans ; Male ; Cartilage, Articular/metabolism* ; Chondrocytes/metabolism* ; Rats, Sprague-Dawley ; Female ; Protective Agents/administration & dosage* ; Single-Cell Analysis ; Middle Aged ; HMGB1 Protein/metabolism*

A combination of the "disease-syndrome-symptom" approach was used to study the syndrome characterization and features of dampness-heat obstruction syndrome in papain-induced knee osteoarthritis(KOA) model rats during the disease process. Forty-eight male SD rats were randomly divided into sham and model groups. The KOA model was established by injecting a mixture of papain and L-cysteine into the joint cavity on days 1, 3, and 5. During the 8 weeks following model establishment, the rats were assessed weekly for the plantar mechanical pain threshold, knee joint diameter, local skin temperature of the knee joint, weight-bearing difference between the two hind feet, and the modified Lequesne MG score of the knee joint. Samples were collected at 1, 2, 4, 6, and 8 weeks after model establishment to observe the gross lesions in cartilage and synovium. Histopathological changes in joint tissues were examined using hematoxylin-eosin, Masson's trichrome, and Senna red O-solid green staining. ELISA and immunohistochemical analysis were performed to detect the levels of interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α, prostaglandin E2(PGE2), and the expression of aquaporins(AQP) 1 and 3 in serum and synovium. The results showed that the ink score of articular cartilage in the model group significantly increased from 4 to 8 weeks, the cartilage Mankin's score and the percentage of Masson-positive area in cartilage increased significantly from 1 to 8 weeks. The percentage of red-stained area for cartilage proteoglycans decreased significantly from 1 to 8 weeks. The synovitis score from 1 to 6 weeks and the percentage of blue-stained collagen fibers in the synovium from 1 to 8 weeks increased significantly, with statistically significant differences compared to the sham group. The mechanical pain threshold in the model group significantly decreased from 1 to 8 weeks, the knee joint diameter significantly increased from 1 to 6 weeks, and the local skin temperature of the knee joint, the weight-bearing difference between the two hind feet, and the modified Lequesne MG score from 1 to 5 weeks significantly increased, all with statistically significant differences compared to the sham group. The levels of IL-1β, IL-6, TNF-α, and PGE2 in serum and synovium of the model group significantly increased from 1 to 6 weeks. Serum TNF-α and PGE2, and synovial IL-1β, also significantly increased at 8 weeks. The levels of cartilage AQP1 and AQP3 significantly increased from 1 to 4 weeks, while synovial AQP1 and AQP3 increased significantly from 1 to 6 weeks, with all differences statistically significant compared to the sham group. In conclusion, papain-induced KOA rats exhibited pathological changes, including articular cartilage degeneration and synovial inflammation, within 1 week of induction. The KOA rats showed characteristics of dampness-heat obstruction syndrome, such as joint pain, swelling, elevated skin temperature, and decreased function, as well as increased inflammatory factors and AQP1、AQP3 in serum and joint tissues within 5 to 6 weeks of disease onset. These results provide an experimental model for studying the syndromes of KOA with dampness-heat obstruction syndrome.
Animals ; Male ; Rats, Sprague-Dawley ; Rats ; Osteoarthritis, Knee/physiopathology* ; Disease Models, Animal ; Humans ; Interleukin-1beta/metabolism* ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Knee Joint/pathology*

This study aims to systematically characterize the targeted effects of Quanduzhong Capsules on cartilage lesions in knee osteoarthritis by integrating spatial transcriptomics data mining and animal experiments validation, thereby elucidating the related molecular mechanisms. A knee osteoarthritis model was established using Sprague-Dawley(SD) rats, via a modified Hulth method. Hematoxylin and eosin(HE) staining was employed to detect knee osteoarthritis-associated pathological changes in knee cartilage. Candidate targets of Quanduzhong Capsules were collected from the HIT 2.0 database, followed by bioinformatics analysis of spatial transcriptomics datasets(GSE254844) from cartilage tissues in clinical knee osteoarthritis patients to identify spatially specific disease genes. Furthermore, a "formula candidate targets-spatially specific genes in cartilage lesions" interaction network was constructed to explore the effects and major mechanisms of Quanduzhong Capsules in distinct cartilage regions. Experimental validation was conducted through immunohistochemistry using animal-derived biospecimens. The results indicated that Quanduzhong Capsules effectively inhibited the degenerative changes in the cartilage of affected joints in rats, which was associated with the regulation of Quanduzhong Capsules on the thioredoxin-interacting protein(TXNIP)-NOD-like receptor family pyrin domain containing 3(NLRP3)-bone morphogenetic protein receptor type 2(BMPR2)-fibronectin 1(FN1)-matrix metallopeptidase 2(MMP2) signal axis in the articular cartilage surface and superficial zones, subsequently inhibiting cartilage matrix degradation leading to oxidative stress and inflammatory diffusion. In summary, this study clarifies the spatially specific targeted effects and protective mechanisms of Quanduzhong Capsules within pathological cartilage regions in knee osteoarthritis, providing theoretical and experimental support for the clinical application of this drug in the targeted therapy on the inflamed cartilage.
Animals ; Osteoarthritis, Knee/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Rats ; Male ; Humans ; Capsules ; Female ; Disease Models, Animal

Objective To investigate the feasibility of breast-conserving surgery(BCS)after neoadjuvant chemotherapy(NC)for triple-negative breast cancer(TNBC).Methods One hundred and thirty-two TNBC patients who were admitted to The Second Affiliated Hospital of Nanchang University from Jun.2014 to Jun.2017 were divided into NC group(62 patients who received NC followed by BCS)and control group(70 patients who received only conventional standard BCS).The response rate was calculated for patients in the NC group.The general clinicopathological data of the 2 groups were compared.The disease-free survival(DFS)of the 2 groups was analyzed using the Kaplan-Meier method,and the effect of NC followed by BCS on the DFS was analyzed using Cox proportional hazards regression method.Results The response rate of patients in the NC group was 96.8%(60/62).There were no differences in age,histological grade,axillary lymph node status or Ki67 index between the 2 groups(all P＞0.05),and the maximum tumor diameter after NC in the NC group was similar to that of the control group(P＞0.05).The ratio of intraventricular cancerous thrombus invasion cases was higher in the NC group than in the control group(P＜0.01).The 1-,3-,and 5-year DFS rates in the NC group were 100%(62 cases),93.5%(58 cases),and 69.4%(43 cases),respectively,with a mean of 55.5 months;the 1-,3-,and 5-year DFS rates in the control group were 100%(70 cases),95.7%(67 cases),and 72.9%(51 cases),respectively,with a mean of 55.6 months;and there was no difference in DFS between the 2 groups(P＞0.05).Cox proportional hazards regression analysis showed that BCS after NC was not a risk factor for DFS in patients with TNBC(hazard ratio=1.133,95%confidence interval 0.600-2.139,P=0.701).Conclusion The response rate to NC is high in TNBC patients,and the treatment strategy of BCS after NC is feasible.

Objective To investigate the effects of scutellarin(SCU)on apoptosis of nucleus pulposus cells and the Janus kinas 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway in intervertebral disc degeneration(IVDD)model rats.Methods IVDD rats were randomly divided into IVDD group,SCU low and high dose groups(SCU-L,SCU-H groups),SCU high dose+JAK2 activator bispecific protein phosphatase 19(DUSP19)group(SCU-H+DUSP19 group)and control group.Enzyme-linked immunosorbent assay(ELISA)method was applied to detect inflammatory factors and oxidative stress level in intervertebral disc tissues.Eosin stai-ning(HE)staining microscopy was used to observe pathological damage of intervertebral disc tissue.Transferase mediated dUTP notch end labeling(TUNEL)staining microscopy was applied to detect apoptosis of nucleus pulpo-sus cells in intervertebral disc tissue.Western blot was applied to detect the expression of JAK2/STAT3 signaling pathway related proteins.Results Animals in the IVDD group were suffered from partial rupture of the fibrous ring in the intervertebral disc tissue,abnormal shrinkage of the nucleus pulposus structure with reduction of nucleus pul-posus cells and blurred boundary with the fibrous ring.The level of TNF-α,IL-1β,ROS and apoptosis rate of nu-cleus pulposus cells,the expression of Bax,cleaved caspase-3,p-JAK2/JAK2,p-STAT3/STAT3 were all elevated and the expression of SOD,type Ⅱ collagen,and polysaccharides decreased(P＜0.05).The morphology,cell structure,density of fibrous ring and nucleus pulposus in the intervertebral disc tissue of the SCU-L and SCU-H groups were improved as compared to IVDD group.The degeneration was alleviated to varying degrees.The level of TNF-α,IL-1β,ROS,apoptosis rate of nucleus pulposus cells and the expression of Bax,cleaved caspase-3,p-JAK2/JAK2,p-STAT3/STAT3 were all reduced and the expression of SOD,type Ⅱ collagen,and polysaccha-rides significantly increased(P＜0.05).DUSP19 partially reverses the effect of SCU on apoptosis of IVDD rats.Conclusions SCU may alleviate apoptosis of nucleus pulposus cells in IVDD rats.

The acupoint sensitization theory,a breakthrough in acupuncture and moxibustion over the past two decades,has greatly enhanced the understanding of meridians and acupoints while increasing the effectiveness of clinical diagnosis and treatment in acupuncture and moxibustion practices.The number of studies on surface sensitization site detection for different diseases is increasing.However,systematic sorting and a summary of the detection mode of surface sensitization points are lacking.Therefore,this study categorizes the method of surface sensitization point detection into two modes:pan-scanning and focused scanning.The classification is based on the characteristics of the openness of the research purpose,the precision of the target range,and the degree of variation during detection.The two modes have considerable differences in the exploration efficiency and the presentation of result.The pan-scanning mode can be further subdivided into holistic and localized pan-scanning modes,whereas the focused-scanning mode can be subdivided into fixed-and variable-focused scanning modes.This study analyzes the application scenarios,characteristics,advantages,and limitations of each detection mode and presents opinions on mode selection,mode innovation,and future development directions.This study aims to provide valuable insights and guidance for the follow-up research on surface sensitization site detection of various diseases.

Background::Oral anti-coagulants (OAC) are the intervention for the prevention of stroke, which consistently improve clinical outcomes and survival among patients with atrial fibrillation (AF). The main purpose of this study is to identify problems in OAC utilization among hospitalized patients with AF in China.Methods::Using data from the Improving Care for Cardiovascular Disease in China-Atrial Fibrillation (CCC-AF) registry, guideline-recommended OAC use in eligible patients was assessed.Results::A total of 52,530 patients with non-valvular AF were enrolled from February 2015 to December 2019, of whom 38,203 were at a high risk of stroke, 9717 were at a moderate risk, and 4610 were at a low risk. On admission, only 20.0% (6075/30,420) of patients with a diagnosed AF and a high risk of stroke were taking OAC. The use of pre-hospital OAC on admission was associated with a lower risk of new-onset ischemic stroke/transient ischemic attack among the diagnosed AF population (adjusted odds ratio: 0.54, 95% confidence interval: 0.43–0.68; P <0.001). At discharge, the prescription rate of OAC was 45.2% (16,757/37,087) in eligible patients with high stroke risk and 60.7% (2778/4578) in eligible patients with low stroke risk. OAC utilization in patients with high stroke risk on admission or at discharge both increased largely over time (all P <0.001). Multivariate analysis showed that OAC utilization at discharge was positively associated with in-hospital rhythm control strategies, including catheter ablation (adjusted odds ratio [OR] 11.63, 95% confidence interval [CI] 10.04–13.47; P <0.001), electronic cardioversion (adjusted OR 2.41, 95% CI 1.65–3.51; P <0.001), and anti-arrhythmic drug use (adjusted OR 1.45, 95% CI 1.38–1.53; P <0.001). Conclusions::In hospitals participated in the CCC-AF project, >70% of AF patients were at a high risk of stroke. Although poor performance on guideline-recommended OAC use was found in this study, over time the CCC-AF project has made progress in stroke prevention in the Chinese AF population.Registration::ClinicalTrials.gov, NCT02309398.

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

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

Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75％(27/36);boys accounted for 64％(23/36).Among the 36 neonates,31(86％)had disease onset within 3 days after birth,and 19(53％)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61％)had left cerebral infarction and 13(36％)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75％)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72％)and seizures in 10 neonates(34％).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33％,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44％(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.