Single-cell transcriptomics of blood identified IFIT1+ neutrophil subcluster expansion in NTM-PD patients.

OBJECTIVE: Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is caused by an imbalance between pathogens and impaired host immune responses. Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are the two major pathogens that cause NTM-PD. In this study, we sought to dissect the transcriptomes of peripheral blood immune cells at the single-cell resolution in NTM-PD patients and explore potential clinical markers for NTM-PD diagnosis and treatment. METHODS: Peripheral blood samples were collected from six NTM-PD patients, including three MAB-PD patients, three MAC-PD patients, and two healthy controls. We employed single-cell RNA sequencing (scRNA-seq) to define the transcriptomic landscape at a single-cell resolution. A comprehensive scRNA-seq analysis was performed, and flow cytometry was conducted to validate the results of scRNA-seq. RESULTS: A total of 27,898 cells were analyzed. Nine T-cells, six mononuclear phagocytes (MPs), and four neutrophil subclusters were defined. During NTM infection, naïve T-cells were reduced, and effector T-cells increased. High cytotoxic activities were shown in T-cells of NTM-PD patients. The proportion of inflammatory and activated MPs subclusters was enriched in NTM-PD patients. Among neutrophil subclusters, an IFIT1+ neutrophil subcluster was expanded in NTM-PD compared to healthy controls. This suggests that IFIT1+ neutrophil subcluster might play an important role in host defense against NTM. Functional enrichment analysis of this subcluster suggested that it is related to interferon response. Cell-cell interaction analysis revealed enhanced CXCL8-CXCR1/2 interactions between the IFIT1+ neutrophil subcluster and NK cells, NKT cells, classical mononuclear phagocytes subcluster 1 (classical Mo1), classical mononuclear phagocytes subcluster 2 (classical Mo2) in NTM-PD patients compared to healthy controls. CONCLUSIONS: Our data revealed disease-specific immune cell subclusters and provided potential new targets of NTM-PD. Specific expansion of IFIT1+ neutrophil subclusters and the CXCL8-CXCR1/2 axis may be involved in the pathogenesis of NTM-PD. These insights may have implications for the diagnosis and treatment of NTM-PD.

Circular RNA circTRAPPC6B Enhances IL-6 and IL-1ß Expression and Repolarizes Mycobacteria Induced Macrophages from M2- to M1-Like Phenotype by Targeting miR-892c-3p.

Mycobacterium tuberculosis (Mtb) infection elicits macrophage polarization into M2 phenotype to block the host's protective immune response. However, it remains unclear how Mtb regulates macrophage polarization. Recent studies have suggested that noncoding RNA may play a role in macrophage polarization. In this study, we investigated the potential involvement of circTRAPPC6B, a circular RNA that is downregulated in tuberculosis (TB) patients, in regulating macrophage polarization. We found that Mtb infection downregulated M1-related IL-6 and IL-1ß while highly expressed M2-related CCL22 and CD163. Overexpressed circTRAPPC6B had switched Mtb-infected macrophages from M2- to M1-like phenotype, accompanied by upregulation of IL-6 and IL-1ß. Meanwhile overexpressed circTRAPPC6B significantly inhibited Mtb growth in macrophages. Our findings suggest that circTRAPPC6B may regulate macrophage polarization by targeting miR-892c-3p, which is highly expressed in TB patients and M2-like macrophages. And miR-892c-3p inhibitor decreased intracellular Mtb growth in macrophages. Thus, TB-inhibited circTRAPPC6B could specifically induce IL-6 and IL-1ß expression to switch/antagonize Mtb-induced macrophage polarization from M2- to M1-like phenotype by targeting miR-892c-3p, leading to enhanced host clearance of Mtb. Our results reveal a potential role for circTRAPPC6B in regulating macrophage polarization during Mtb infection and provide new insights into the molecular mechanisms underlying host defense against Mtb.

Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA-874-3p.

OBJECTIVES: Genetic and epigenetic mechanisms regulate antimicrobial immunity against Mycobacterium tuberculosis (Mtb) infection. METHODS: The present study assessed circular RNA TRAPPC6B (circTRAPPC6B) for antimicrobial immune functions and defined mechanisms wherein circTRAPPC6B regulates Mtb growth, autophagy and microRNA in macrophages. RESULTS: The Mtb infection of monocytes/macrophages resulted in a significantly decreased level of circTRAPPC6B that inhibited intracellular Mtb growth in macrophages. Conversely, circTRAPPC6B expression enhanced autophagy or autophagy-associated protein LC3-II production in Mtb-infected macrophages. circTRAPPC6B-enhanced autophagy aggregation or sequestration was also observed in fluorescence in situ hybridisation (FISH) analysis and confocal imaging. Mechanistically, circTRAPPC6B targets an inhibiting element miR-874-3p, as shown by bioinformatics, dual-luciferase reporter gene analysis and pull-down assay, respectively. Notably, miR-874-3p prohibited autophagy via suppressing autophagy protein ATG16L1 by binding to its 3'-untranslated region (UTR) in Mtb-infected macrophages and thus promoting intracellular Mtb growth. Concurrently, circTRAPPC6B enhanced autophagy in Mtb-infected macrophages by blocking the ability of miR-874-3p to inhibit ATG16L1. Thus, circTRAPPC6B antagonises the ability of miR-874-3p to suppress ATG16L1 expression and activate and enhance autophagy sequestration to restrict Mtb growth in macrophages. CONCLUSION: The current findings suggested that both circTRAPPC6B and miR-874-3p mechanisms can be explored as potential therapeutics against Mtb infection.

["Multi-component-multi-target-multi-pathway" mechanism of Kuihua Hugan Tablets based on network pharmacology].

To predict the targets of active ingredients of Kuihua Hugan Tablets by network pharmacology, and explore the "multi-component-multi-target-multi-pathway" hepatoprotective mechanism of action. First, through traditional Chinese medicine systems pharmacology(TCMSP) and TCM Database@Taiwan Database, main active ingredients of Kuihua Hugan Tablets were screened out based on oral bioavailability(OB), drug-likeness(DL) and effective half-lives(HL). The targets of active ingredients of Kuihua Hugan Tablets were predicted based on the PharmMapper method. Then, the prediction was conducted by screening the target genes associated with chronic hepatitis and early cirrhosis through CooLGeN and GeneCards databases. Target gene functions and signal pathways were analyzed by bioinformatics annotation database Metascape. Cytoscape software was used to construct the Kuihua Hugan Tablets ingredient-target and ingredient-target-pathway network. String database combined with Cytoscape software was used to construct the networks of component-target and component-target-pathway. STRING database was combined with Cytoscape software to draw protein-protein interaction(PPI) network and conduct network topology analysis. Finally, Systems Dock Web Site software was applied in verifying the molecular docking between active ingredients and potential protein targets. A total of 26 compounds and 509 potential targets were screened out from Kuihua Hugan Tablets in the experiment. The results of PPI network analysis indicated that albumin(ALB), insulin-like growth factor 1(IGF1), matrix metalloproteinase-9(MMP9), matrix metalloproteinase-2(MMP2), non-receptor tyrosine kinase proto-oncogene(SRC), estrogen receptor 1(ESR1) and cancer-signal transduction-inflammation-drugs metabolism-related biological processes and metabolic pathways were closely associated with the active ingredients in Kuihua Hugan Tablets. The effects of Kuihua Hugan Tablets in alleviating chronic hepatitis and early cirrhosis indicated the multi-component, multi-target, and multi-pathway characteristics of traditional Chinese medicines, providing new ideas for further research and development of Kuihua Hugan Tablets.

Protective Effect of Norcantharidin on Collagen-Induced Arthritis Rats.

OBJECTIVE: To observe the effect of norcantharidin (NCTD) on collagen-induced arthritis (CIA) rats. METHODS: Sixty Sprague-Dawley(SD) rats were randomly divided into 6 groups (n=10): normal group, CIA model group(model group), NCTD low-dose group [1.35 mg/(kg•d)], NCTD middle-dose group [2.7 mg/(kg•d)], NCTD high-dose group [5.4 mg/(kg•d)] and methotrexate (MTX) group [1.8 mg/(kg/w)]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin (H&E) staining. The serum levels of interleukin (IL) 1ß, IL-6, tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), IL-17 and transform growth factor (TGF) ß were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expression of retinoid-related orphan nuclear receptorγt (RORγt) and forkhead box P3 (Foxp3) in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction. RESULTS: MTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats' ankle joints compared with the model group (P<0.05 or P<0.01). All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats (P<0.05). Only middle- and high-dose of NCTD prominently decreased serum IL-1ß and TGF-ß levels of CIA rats (P<0.05). However, NCTD has no effect on vascular endothelial growth factor (VEGF) level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group (P<0.05). The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group (P<0.05). CONCLUSIONS: NCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.

[Protective effect of acupuncture on heart in mice with hyperlipemia and its mechanism].

OBJECTIVE: To observe the inhibiting effect of acupuncture on blood lipid, myocardial hypertrophy and fibrosis in mice with hyperlipemia, and explore its possible action mechanism. METHODS: Ten inbred mice (C57) were applied. Forty ApoE(-/-) mice who removed gene of apolipoprotein E were randomly divided into a control group, a non-acupoint group, an acupoint group and a medication group. The points 0. 5 cm and 1 cm next to the end of mice tail were respectively punctured in the non-acupoint group; "Neiguan" (PC 6) and "Fenglong" (ST 40) were punctured in the acupoint group; intragastric administration of simvastatin was applied in the medication group. After 8 weeks of treatment, the changes of total cholesterol (TC) and ratio of heart to body mass in each group were measured; changes of cardiac muscle fiber and ventricular wall thickness were observed; enzyme linked immunosorbent assay (ELISA) was used to test the level of angiotensin II (Ang I ) in plasma, and western blotting method was used to test protein content of angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) in the heart. RESULTS: After 8 weeks of intervention, compared with the control group, rising range of blood lipid was obviously decreased (P<0.01) in the acupoint group and medication group, ratio of P<0.01), myocardial heart to body mass was decreased (P<0.05), thickness of ventricular wall was reduced (P fibrosis was relieved, levels of Ang II and ET-1 in plasma were decreased (P<0. 05), content of NO was increased (P<0. 05), and protein content of AT1R and ETAR was decreased in the heart (P<0. 05). CONCLUSION: 40) could inhibit the rising of blood lipid in ApoE(-/-) mice, lower the levels of Ang II and ET-1 in peripheral blood, increase the content of NO and inhibit the expression of AT1R and ETAR in heart tissue, which could relieve myocardial hypertrophy and fibrosis to play a protective role on heart.

Effects of cold-damp and hot-damp environment on VEGF and IL-1 expression in joint cartilage cells in adjuvant arthritis in rats.

OBJECTIVE: To study the effects of environmental factors on the degree of injury and expression of vascular endothelial growth factor (VEGF) and interleukin-1 (IL-1) in cartilage cells of the joint in a rat model of adjuvant arthritis (AA). METHODS: SD rats aged 10 months were randomly divided into 4 groups that varied by temperature and humidity housing conditions and induction of AA: a control group, a model group, a cold-damp group, and a hot-damp group. All groups except the control group were induced with AA. After 4 w, VEGF and IL-1 expression in cartilage cells of ankle joints of hind limbs were observed. RESULTS: Mean area, optical density, and numbers of VEGF- and IL-1-positive cells in the model group, the cold-damp group, and the hot-damp group were significantly higher than that of the control group (all P < 0.05). Optical density and positive cell numbers in the cold-damp group and the hot-damp group were significantly higher than that of the model group (all P < 0.05). Optical density and positive cell numbers in the hot-damp group were significantly higher than that of the cold-damp group. Bone in the hot-damp and cold-damp groups was severely injured. CONCLUSION: Environmental factors such as high humidity combined with either high or low temperature increase the severity of damage and expression of VEGF and IL-1 in cartilage cells of joints in rats induced with AA.

Assessment of clinical effect of therapy combining disease with syndrome on rheumatoid arthritis.

OBJECTIVE: To observe the effectiveness and safety of a therapy combining disease with syndrome on rheumatoid arthritis. METHODS: Eighty patients with rheumatoid arthritis belonging to syndrome of damp-heat obstruction were randomly divided into a treatment group and a control group according to stratified blocked randomization method. Forty cases in the control group orally took Loxoprofen Sodium Tablet and Leifumite Tablet and the other 40 cases in the treatment group orally took a Chinese medicine for 12 weeks as a course of treatment. ACR therapeutic effect was used as the standard for evaluating the total therapeutic effect. RESULTS: After 12 weeks of treatment, there was a statistical difference (P < 0.01) in the improvement of VAS score, morning stiffness time, number of swelling joints, index of swelling joints, number of joints with tenderness, index of joints with tenderness, average grip strength of both hands, DSA28 score, HAQ, patient's assessment, physician's assessment, ESR, CRP and RF in both groups. The improvement of morning stiffness time, number of swelling joints, index of swelling joints, grip strength, HAQ and patient's assessment in the treatment group was much better than that in the control groups with statistical difference (P < 0.05). ACR20, ACR50 and ACR70 was 27.5% (11/40), 37.5% (15/40) and 22.5% (9/40) respectively in the treatment group and 40% (16/40), 27.5% (11/42) and 10.0% (4/40) respectively in the control group with statistical difference (P < 0.05) in the superiority of the treatment group over the control group. The incidence of adverse reaction in the control group was higher than that in the treatment group (P < 0.05). CONCLUSION: Definite therapeutic effect and high safety can be achieved in using the therapy combining disease with syndrome to treat rheumatoid arthritis belonging to syndrome of damp-heat obstruction.