Dataset Information

Du-Huo-Ji-Sheng-Tang Attenuates Inflammation of TNF-Tg Mice Related to Promoting Lymphatic Drainage Function.

ABSTRACT: To investigate whether Du-Huo-Ji-Sheng-Tang (DHJST) attenuate inflammation of RA related to lymphatic drainage function in vivo, we treated eight 3-month-old TNF-Tg mice with DHJST (12?g/kg) or the same volume of physiological saline once every day for 12 weeks, and 3-month-old WT littermates were used as negative control. After twelve weeks, we performed NIR-ICG imaging and found that DHJST increased the ICG clearance at the footpad and the pulse of efferent lymphatic vessel between popliteal lymph node and footpad. Histology staining at ankle joints showed that DHJST decreases synovial inflammation, bone erosion, cartilage erosion, and TRAP+ osteoclast area in TNF-Tg mice. Immunohistochemical staining by using anti-Lyve-1 and anti-podoplanin antibody showed that DHJST stimulated lymphangiogenesis in ankle joints of TNF-Tg mice. And zebrafish study suggested that DHJST promoted the formation of lymphatic thoracic duct. In conclusion, DHJST inhibits inflammation severity and promotes lymphangiogenesis and lymphatic drainage function of TNF-Tg mice.

SUBMITTER: Chen Y

PROVIDER: S-EPMC4863122 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Du-Huo-Ji-Sheng-Tang Attenuates Inflammation of TNF-Tg Mice Related to Promoting Lymphatic Drainage Function.

Chen Yan Y Li Jinlong J Li Qiang Q Wang Tengteng T Xing Lianping L Xu Hao H Wang Yongjun Y Shi Qi Q Zhou Quan Q Liang Qianqian Q

Evidence-based complementary and alternative medicine : eCAM 20160427

To investigate whether Du-Huo-Ji-Sheng-Tang (DHJST) attenuate inflammation of RA related to lymphatic drainage function in vivo, we treated eight 3-month-old TNF-Tg mice with DHJST (12 g/kg) or the same volume of physiological saline once every day for 12 weeks, and 3-month-old WT littermates were used as negative control. After twelve weeks, we performed NIR-ICG imaging and found that DHJST increased the ICG clearance at the footpad and the pulse of efferent lymphatic vessel between popliteal l ...[more]

PMID: 27239212

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Project description:Osteoarthritis (OA) is a chronic degenerative joint disease characterized by the breakdown of articular cartilage, subchondral bone remodeling, and inflammation of the synovium. In this study, we investigated whether Fang-Ji-Huang-Qi-Tang (FJHQT) decoction improved the joint structure of OA or delayed the process of knee joint degeneration in OA mice by promoting lymphatic drain function. The mice were randomly divided into four groups, the sham group, the PBS group, the FJHQT-treated group, and the Mobic-treated group. The mice in each group were tested for lymphatic draining function at 4, 6, 8, and 10 weeks postsurgery (WPS), then sacrificed (N?=?10/group). Using a near-infrared indocyanine green (NIR-ICG) lymphatic imaging system, we found that the lymphatic drain function was significantly reduced in the PBS group compared with the sham group. After treatment with the FJHQT decoction, the lymphatic draining function improved at 4 wps and 6 wps. The results of the analysis indicated a strong correlation between lymphatic draining function (ICG clearance) and the degree of joint structural damage (OARSI score). By Alcian blue/orange G (ABOG) staining of the paraffin sections, the FJHQT-treated group exhibited less cartilage destruction and lower OARSI scores. Moreover, the result of immunohistochemical staining (IHC) shows that FJHQT decoction increased the content of type II collagen in knee joints of OA mice at 4 wps and 6 wps. By the double immunofluorescence staining of podoplanin and smooth muscle actin in the paraffin sections, the capillaries and mature lymphatics in the FJHQT group increased at 4 wps. In conclusion, the FJHQT decoction can increase lymphatic vessel number, promote joint lymphatic draining function, and postpone knee osteoarthritis pathologic progression in the early stage of a collagen-induced mouse model. Therefore, the application of sufficient lymphatic drainage in the knee joint may be a new treatment method for knee joint osteoarthritis (KOA).

Project description:While rheumatoid arthritis patients and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis display lymphatic drainage deficits, the mechanisms responsible remain unknown. As ultrastructural studies of joint-draining popliteal lymphatic vessels (PLVs) in TNF-Tg mice revealed evidence of lymphatic muscle cell (LMC) damage, we aimed to evaluate PLV-LMC coverage in TNF-Tg mice. We tested the hypothesis that alpha smooth muscle actin (αSMA)+ PLV-LMC coverage decreases with severe inflammatory-erosive arthritis, and is recovered by anti-TNF therapy facilitated by increased PLV-LMC turnover during amelioration of joint disease. TNF-Tg mice with established disease received anti-TNF monoclonal antibody (mAb) or placebo IgG isotype control mAb therapy (n = 5) for 6-weeks, while wild-type (WT) littermates (n = 8) received vehicle (PBS). Bromodeoxyuridine (BrdU) was also administered daily during the treatment period to monitor PLV-LMC turnover. Effective anti-TNF therapy was confirmed by longitudinal assessment of popliteal lymph node (PLN) volume via ultrasound, PLV contraction frequency via near-infrared imaging of indocyanine green, and ankle bone volumes via micro-computed tomography (micro-CT). Terminal knee micro-CT, and ankle and knee histology were also performed. PLVs were immunostained for αSMA and BrdU to evaluate PLV-LMC coverage and turnover, respectively, via whole-mount fluorescent microscopy. Anti-TNF therapy reduced PLN volume, increased talus and patella bone volumes, and reduced tarsal and knee synovial areas compared to placebo treated TNF-Tg mice (p < 0.05), as expected. Anti-TNF therapy also increased PLV contraction frequency at 3-weeks (from 0.81 ± 1.0 to 3.2 ± 2.0 contractions per minute, p < 0.05). However, both anti-TNF and placebo treated TNF-Tg mice exhibited significantly reduced αSMA+ PLV-LMC coverage compared to WT (p < 0.05). There was no correlation of αSMA+ PLV-LMC coverage restoration with amelioration of inflammatory-erosive arthritis. Similarly, there was no difference in PLV-LMC turnover measured by BrdU labeling between WT, TNF-Tg placebo, and TNF-Tg anti-TNF groups with an average of < 1% BrdU+ PLV-LMCs incorporated per week. Taken together these results demonstrate that PLV-LMC turnover in adult mice is limited, and that recovery of PLV function during amelioration of inflammatory-erosive arthritis occurs without restoration of αSMA+ LMC coverage. Future studies are warranted to investigate the direct and indirect effects of chronic TNF exposure, and the role of proximal inflammatory cells on PLV contractility.

Project description:BackgroundLymphatic dysfunction exists in tumor necrosis factor transgenic (TNF-Tg) mice and rheumatoid arthritis (RA) patients. While joint-draining TNF-Tg popliteal lymphatic vessels (PLVs) have deficits in contractility during end-stage arthritis, the nature of lymphatic muscle cells (LMCs) and their TNF-altered transcriptome remain unknown. Thus, we performed single-cell RNA-sequencing (scRNAseq) on TNF-Tg LMCs in PLVs efferent to inflamed joints versus wild-type (WT) controls.MethodsSingle-cell suspensions of PLVs were sorted for smooth muscle cells (SMCs), which was validated by Cspg4-Cre;tdTomato reporter gene expression. Single-cell RNA-seq was performed on a 10x Genomics platform and analyzed using the Seurat R package. Uniform Manifold Approximation and Projections (UMAPs) and Ingenuity Pathway Analysis software were used to assess cell clusters and functional genomics in WT vs. TNF-Tg populations.ResultsFluorescent imaging of Cspg4-Cre;tdTomato vessels demonstrated dim PLVs and strong reporter gene expression in the adjacent superficial saphenous vein, which was corroborated by flow cytometry of LMCs and vascular smooth muscle cells (VSMCs) from these vessels. Due to their unique morphology, these populations could also be readily detected by scatter analysis of cells from non-fluorescent mice. Bioinformatics analysis of flow sorted WT and TNF-Tg cells identified 20 unique cell clusters that together were 22.4% LMCs, 15.0% VSMCs, and 62.6% non-muscle cells of 8879 total cells. LMCs and M2-macrophages were decreased, while inflammatory monocytes were increased in TNF-Tg lower limb vasculature. SMC populations were defined by Cald1, Tpm1, and Pdgfrb expression and were enriched in myofibroblast-like gene expression. TNF-Tg LMCs exhibited enhanced functional genomics associated with cell death, phagocyte recruitment, and joint inflammation. Among the most prominent TNF-induced genes in SMCs were Mmp3, Cxcl12, and Ccl19, and the most downregulated genes were Zbtb16, Galnt15, and Apod.ConclusionsSingle-cell RNA-seq can be used to investigate functional genomics of lower limb vasculature in mice. Our findings confirm the inflammatory transcriptome of TNF-Tg vessels and altered gene expression in SMC populations. This study further supports a potential role of mesenchymal stromal cells in inflammatory-erosive arthritis pathogenesis, and warrants future studies to define the effects of this TNF-altered transcriptome on PLV function and joint homeostasis.

Dataset Information

Du-Huo-Ji-Sheng-Tang Attenuates Inflammation of TNF-Tg Mice Related to Promoting Lymphatic Drainage Function.

Publications

Du-Huo-Ji-Sheng-Tang Attenuates Inflammation of TNF-Tg Mice Related to Promoting Lymphatic Drainage Function.

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