OBJECTIVE: To evaluate the dynamic changes of glucocorticoid (GC) dose and the feasibility of GC discontinuation in rheumatoid arthritis (RA) patients under the background of Chinese medicine (CM). METHODS: This multicenter retrospective cohort study included 1,196 RA patients enrolled in the China Rheumatoid Arthritis Registry of Patients with Chinese Medicine (CERTAIN) from September 1, 2019 to December 4, 2023, who initiated GC therapy. Participants were divided into the Western medicine (WM) and integrative medicine (IM, combination of CM and WM) groups based on medication regimen. Follow-up was performed at least every 3 months to assess dynamic changes in GC dose. Changes in GC dose were analyzed by generalized estimator equation, the probability of GC discontinuation was assessed using Kaplan-Meier curve, and predictors of GC discontinuation were analyzed by Cox regression. Patients with <12 months of follow-up were excluded for the sensitivity analysis. RESULTS: Among 1,196 patients (85.4% female; median age 56.4 years), 880 (73.6%) received IM. Over a median 12-month follow-up, 34.3% (410 cases) discontinued GC, with significantly higher rates in the IM group (40.8% vs. 16.1% in WM; P<0.05). GC dose declined progressively, with IM patients demonstrating faster reductions (median 3.75 mg vs. 5.00 mg in WM at 12 months; P<0.05). Multivariate Cox analysis identified age <60 years [P<0.001, hazard ratios (HR)=2.142, 95% confidence interval (CI): 1.523-3.012], IM therapy (P=0.001, HR=2.175, 95% CI: 1.369-3.456), baseline GC dose ⩽7.5 mg (P=0.003, HR=1.637, 95% CI: 1.177-2.275), and absence of non-steroidal anti-inflammatory drugs use (P=0.001, HR=2.546, 95% CI: 1.432-4.527) as significant predictors of GC discontinuation. Sensitivity analysis (545 cases) confirmed these findings. CONCLUSIONS RA patients receiving CM face difficulties in following guideline-recommended GC discontinuation protocols. IM can promote GC discontinuation and is a promising strategy to reduce GC dependency in RA management. (Trial registration: ClinicalTrials.gov, No. NCT05219214).
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Arthritis, Rheumatoid/drug therapy* ; Glucocorticoids/therapeutic use* ; Medicine, Chinese Traditional ; Retrospective Studies

Metachromatic leukodystrophy (MLD) is an inherited disease caused by a deficiency of the enzyme arylsulfatase A (ARSA). Lentivirus-modified autologous hematopoietic stem cell gene therapy (HSCGT) has recently been approved for clinical use in pre and early symptomatic children with MLD to increase ARSA activity. Unfortunately, this advanced therapy is not available for most patients with MLD who have progressed to more advanced symptomatic stages at diagnosis. Patients with late-onset juvenile MLD typically present with a slower neurological progression of symptoms and represent a significant burden to the economy and healthcare system, whereas those with early onset infantile MLD die within a few years of symptom onset. We conducted a pilot study to determine the safety and benefit of HSCGT in patients with postsymptomatic juvenile MLD and report preliminary results. The safety profile of HSCGT was favorable in this long-term follow-up over 9 years. The most common adverse events (AEs) within 2 months of HSCGT were related to busulfan conditioning, and all AEs resolved. No HSCGT-related AEs and no evidence of distorted hematopoietic differentiation during long-term follow-up for up to 9.6 years. Importantly, to date, patients have maintained remarkably improved ARSA activity with a stable disease state, including increased Functional Independence Measure (FIM) score and decreased magnetic resonance imaging (MRI) lesion score. This long-term follow-up pilot study suggests that HSCGT is safe and provides clinical benefit to patients with postsymptomatic juvenile MLD.
Humans ; Leukodystrophy, Metachromatic/genetics* ; Pilot Projects ; Genetic Therapy/methods* ; Hematopoietic Stem Cell Transplantation ; Male ; Follow-Up Studies ; Female ; Lentivirus/genetics* ; Child ; Child, Preschool ; Hematopoietic Stem Cells/metabolism* ; Cerebroside-Sulfatase/metabolism* ; Adolescent

Acidosis, regardless of hypoxia involvement, is recognized as a chronic and harsh tumor microenvironment (TME) that educates malignant cells to thrive and metastasize. Although overwhelming evidence supports an acidic environment as a driver or ubiquitous hallmark of cancer progression, the unrevealed core mechanisms underlying the direct effect of acidification on tumorigenesis have hindered the discovery of novel therapeutic targets and clinical therapy. Here, chemical-induced and transgenic mouse models for colon, liver and lung cancer were established, respectively. miR-7 and TGF-β2 expressions were examined in clinical tissues (n = 184). RNA-seq, miRNA-seq, proteomics, biosynthesis analyses and functional studies were performed to validate the mechanisms involved in the acidic TME-induced lung cancer metastasis. Our data show that lung cancer is sensitive to the increased acidification of TME, and acidic TME-induced lung cancer metastasis via inhibition of miR-7-5p. TGF-β2 is a direct target of miR-7-5p. The reduced expression of miR-7-5p subsequently increases the expression of TGF-β2 which enhances the metastatic potential of the lung cancer. Indeed, overexpression of miR-7-5p reduces the acidic pH-enhanced lung cancer metastasis. Furthermore, the human lung tumor samples also show a reduced miR-7-5p expression but an elevated level of activated TGF-β2; the expressions of both miR-7-5p and TGF-β2 are correlated with patients' survival. We are the first to identify the role of the miR-7/TGF-β2 axis in acidic pH-enhanced lung cancer metastasis. Our study not only delineates how acidification directly affects tumorigenesis, but also suggests miR-7 is a novel reliable biomarker for acidic TME and a novel therapeutic target for non-small cell lung cancer (NSCLC) treatment. Our study opens an avenue to explore the pH-sensitive subcellular components as novel therapeutic targets for cancer treatment.

AIM:To observe the clinical effect of ranibizumab combined with 577nm micropulse laser in the treatment of severe diabetic macular edema(DME). METHODS:There were 52 eyes of 52 patients diagnosed with severe DME who admitted to the People's Hospital of Guangxi Zhuang Autonomous Region from June 2016 to September 2019. The patients were randomly divided into the observation group(26 patients with 26 eyes, treated with ranibizumab combined with 577nm micropulse laser)and the control group(26 patients with 26 eyes, treated with ranibizumab alone). Patients in both groups received intravitreal injection of ranibizumab with “3+PRN” regimen. Followed up at 9mo after treatment to observe the central macular thickness(CMT), the best corrected visual acuity(BCVA)and the times of intravitreal injection of ranibizumab in the two groups.RESULTS:Compared with before treatment, the CMT and BCVA of the two groups were significantly improved at each time point after treatment(all P<0.001), but there was no difference between the two groups(P>0.05). During the follow-up period, the times of vitreous injection of ranibizumabin the observation group was significantly less than that in the control group(5.88±1.24 times vs 7.12±1.24 times, P=0.001). CONCLUSION:Both ranibizumab combined with 577nm micropulse laser and ranibizumab alone are effective in reducing edema and improving vision in patients with severe DME, but the combination therapy reduces the times of injection.

In this study, UPLC-Q-Exactive-MS/MS was used to rapidly analyze the chemical constituents of Meconopsis quintupli-nervia, and the anti-liver fibrosis mechanism of M. quintuplinervia was preliminarily analyzed by network pharmacology, molecular docking, and cell experiments. The chemical constituents of M. quintuplinervia were identified according to the information of MS~1 and MS~2, as well as the data in the literature and databases. SwissTargetPrediction and TargetNet were used to predict the potential targets. The targets related to liver fibrosis were collected from GeneCards and OMIM. The protein-protein interaction(PPI) network was constructed by STRING. Cytoscape 3.6.1 was used to construct and analyze the "constituent-target-disease" network to obtain key targets and their corresponding constituents in the network. DAVID 6.8 was used for GO analysis and KEGG signaling pathway enrichment analysis. Finally, the preliminary verification was carried out by molecular docking and cell experiments. As a result, 106 chemical constituents were identified from M. quintuplinervia, including 66 flavonoids, 16 alkaloids, 18 phenolic acids, 1 anthocyanin, and 5 other constituents. Among them, 3 constituents were identified as potential new compounds, and 59 constituents were reported in M. quintuplinervia for the first time. Network pharmacology analysis showed that M. quintuplinervia presumably acted on AKT1, SRC, JUN, EGFR, STAT3, HSP90 AA1, MAPK3, and other core targets through luteolin, isorhamnetin, quercetin, apigenin, kaempferide, amurine, 2-methylflavinantine, allocryptopine, the multi and other active compounds, thereby regulating the PI3 K/AKT signaling pathway, pathways in cancer, proteoglycans in cancer, FoxO signaling pathway, and other pathways to exert anti-liver fibrosis effects. M. quintuplinervia extract(MQE) could significantly down-regulate PI3 K and AKT protein levels in the HSC-T6 cell model induced by TGF-β1, suggesting that MQE may have the ability to regulate the PI3 K/AKT signaling pathway. The findings of this study indicated that the anti-liver fibrosis effect of M. quintuplinervia had multi-constituent, multi-target, and multi-pathway characteristics, which may provide a scientific basis for the research on the pharmacodynamic materials, action mechanism, and quality markers of M. quintupli-nervia.
Tandem Mass Spectrometry ; Molecular Docking Simulation ; Network Pharmacology ; Proto-Oncogene Proteins c-akt ; Papaveraceae ; Liver Cirrhosis ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVE: In order to conduct high-throughput genome-wide translocation sequencing based on CRISPR/Cas9, Nalm6-cas9 monoclonal cell line expressing Cas9 protein was constructed by lentivirus transduction. METHODS: Lentiviral vectors LentiCas9-Blast, pSPAX2, and pMD2.G were used to co-transfect HEK293T cells to obtain recombinant lentivirus. After Nalm6 cells were infected with the recombinant lentivirus, the cells were screened by Blasticidin, and multiple monoclonal cell lines expressing Cas9 protein were obtained by limited dilution. Western blot was used to detect the expression level of Cas9 protein in monoclonal cell lines, and cell count analysis was used to detect the proliferation activity of monoclonal cell lines. LentiCRISPRV2GFP-Δcas9, LentiCRISPRV2GFP-Δcas9-AF4, LentiCRISPRV2GFP-Δ cas9-MLL plasmids were constructed, and transfected with pSPAX2 and pMD2.G, respectively. T vector cloning was used to detect the function of Cas9 protein in Nalm6-Cas9 monoclonal cell line infected with virus. RESULTS: Western blot showed that Nalm6-Cas9_1-6 monoclonal cell line had high expression of Cas9 protein. Cell count analysis showed that high expression of Cas9 protein in Nalm6-Cas9_1-6 monoclonal cell line did not affect cell proliferation activity. The Nalm6-Cas9_1-6 monoclonal cell line had high cleavage activity, and the editing efficiency of AF4 and MLL genes was more than 90% which was determined by T vector cloning. CONCLUSION Nalm6-Cas9_1-6 monoclonal cell line stably expressing highly active Cas9 protein was obtained, which provided a basis for exploring the translocation of MLL in therapy-related leukemias based on CRISPR/Cas9 genome-wide high-throughput genome-wide translocation sequencing.
CRISPR-Associated Protein 9/genetics* ; CRISPR-Cas Systems ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus/genetics* ; Plasmids

Objective To explore the joint contact force, ligament tensile force and force transmission mode of foot internal structure in Down’s syndrome child (DSC) during standing. Methods The finite element models of foot were constructed based on CT image data from one DSC and one typically developing child (TDC). The models were validated by plantar pressure measurement during static standing. To simulate foot force during standing, the ground reaction force and the triceps surae force were applied as the loading condition. Contact pressure of the tibiotalar, talonavicular and calcaneocuboid joints, tensile force of the spring and plantar calcaneocuboid ligaments, and force transmission mode in transverse tarsal joints were calculated and analyzed. Results The finite element models of foot were validated to be reliable. Compared with the TDC, the DSC showed higher contact pressure at the tibiotalar joint and lower contact pressure at the talonavicular joint. The tensile force of spring and plantar calcaneocuboid ligaments of DSC was 10 times and 58 times of TDC, respectively. The forces transmitted through both mediate and lateral columns in DSC were lower than those in TDC. Conclusions Abnormal contact pressure of the tibiotalar joint, larger tensile force of midfoot ligaments and smaller force of the transverse tarsal joint were found in DSC during standing. The abnormal alteration of stress patterns in foot internal structure of DSC should be fully considered in clinical rehabilitation, so as to provide theoretical references for screening and making intervention plans for early rehabilitation, as well as designing individualized orthopedic insoles.

OBJECTIVE: To study the expression of B lymphocyte-induced mature protein-1 (BLIMP-1) in regulatory T cells (Tregs) of children with aplastic anemia (AA), and analyze its correlation with the number of Tregs and the levels of inhibitory cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β in plasma. METHODS: The peripheral blood samples of 10 newly diagnosed AA children and 10 healthy children were collected for experiment. qPCR was used to detect FOXP3 and PRDM1 mRNA expression levels. Flow cytometry was used to detect the proportion of Tregs, the expression of BLIMP-1 in Tregs, and the levels of cytokines such as IL-2, IL-17A, IL-6, interferon (IFN)-γ, IL-10 and TGF-β in plasma. Pearson correlation model was used to evaluate the relationship between the expression of BLIMP-1 in Treg and the number of Tregs, as well as the levels of IL-10 and TGF-β in plasma. RESULTS: Compared with control group, the proportion of Tregs in peripheral blood of AA children was decreased significantly (P<0.001); The plasma levels of proinflammatory cytokines IL-2, IL-6 and IFN-γ in AA children were increased significantly (P=0.033, P=0.031, P=0.006), and IL-17A also was increased but the difference was not statistically significant (P=0.052), while anti-inflammatory cytokines IL-10 and TGF-β were significantly reduced (P=0.048, P=0.002). The relative expressions level of FOXP3 and PRDM1 mRNA in AA children were significantly lower than those in control group (P=0.037, P=0.016). The expression of BLIMP-1 protein in Tregs of AA children was significantly lower than that in control group (P<0.001). The expression level of BLIMP-1 protein in Tregs was positively correlated with the percentage of Tregs in lymphocytes (r=0.671, P=0.001), and was also positively correlated with the levels of IL-10 and TGF-β in plasma (r=0.500, P=0.029; r=0.486, P=0.030). CONCLUSION The expression of BLIMP-1 in Tregs of AA children is impaired, and the low expression of BLIMP-1 is related to the decrease of the number in Tregs and IL-10 and TGF-β expressions.
Anemia, Aplastic ; Child ; Cytokines ; Flow Cytometry ; Forkhead Transcription Factors ; Humans ; Positive Regulatory Domain I-Binding Factor 1 ; T-Lymphocytes, Regulatory ; Transforming Growth Factor beta

Objective:To develop a patient-reported outcomes (PRO) scale for post-stroke aphasia based on the Traditional Chinese Medicine (TCM) holism. Methods:Referring to the TCM holism, the theoretical model was established following the standard process for the development of the PRO scale. An item pool was established and optimized with case review, patient interview, expert questionnaire survey and consensus conference. The PRO scale was established finally. Results:A pool of post-stroke aphasia items based on the holistic view of TCM was constructed in the physical, psychological and social model. The Expert Comment Form for the PRO Scale for Aphasia after Stroke was formed after sorting out the item pool. After expert surveys and meeting discussion, 40 items were finally selected to form the first version of Patient-reported Outcomes Scale for Post-stroke Aphasia-TCM. Conclusion:The first version of Patient-reported Outcomes Scale for Post-stroke Aphasia-TCM has been developed based on the TCM holism and the concept of PRO, which can be evaluated clinically.

Objective:To evaluate the role of tumour necrosis factor-α-induced protein 8-like 2 (TIPE2) in the acute lung injury (ALI) induced by endotoxin in mice.Methods:Forty SPF healthy adult male BALB/c mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=10 each) using a random number table method: vehicle plasmid group (VP group), vehicle plasmid plus ALI group (VP+ ALI group), TIPE2 adeno-associated virus overexpression group (T group) and TIPE2 adeno-associated virus overexpression plus ALI group (T+ ALI group). The mice in VP and VP+ ALI groups were injected with empty adeno-associated virus, while the mice in T and T+ ALI groups were intratracheally given adeno-associated virus carrying TIPE interference sequence.Three weeks later, the model of endotoxin-induced ALI was established.Lipopolysaccharide (LPS) 5 mg/kg was intratracheally given in VP+ ALI and T+ ALI groups, and the equal volume of phosphate buffered saline (PBS) was given in VP and T groups.Blood samples were obtained from the abdominal aorta at 24 h after injection of LPS for blood gas analysis, oxygenation index (OI) was calculated, and tumor necrosis factor-alpha (TNF-α) in serum were detected by enzyme-linked immunosorbent assay.The animals were then sacrificed, and lung tissues were removed for examination of pathological changes which were scored after haematoxylin and eosin staining, for calculation of the wet/dry weight ratio (W/D ratio) and for determination of myeloperoxidase (MPO) activity and the expression of TIPE2, phosphorylated c-Jun N-terminal kinase (p-JNK) and nuclear factor kappa B(NF-κB) (by Western blot). Results:Compared with VP group, the lung injury score, W/D ratio, MPO activity and concentration of serum TNF-α were significantly increased, PaO 2 and OI were decreased, expression of TIPE2 was down-regulated and expression of p-JNK and NF-κB was up-regulated in VP+ ALI group ( P<0.05). Compared with VP+ ALI group, the lung injury score, W/D ratio, MPO activity and concentration of serum TNF-α were significantly decreased, PaO 2 and OI were increased, expression of TIPE2 was up-regulated and expression of p-JNK and NF-κB was down-regulated in T+ ALI group ( P<0.05). Conclusion:The down-regulation of TIPE2 expression is involved in the process of ALI induced by endotoxin in mice.