Construction of a novel immune-related prognostic-predicting model of gastric cancer.

Gastric cancer (GC) is a common primary stomach tumor of the central nervous system with a poor prognosis. In this study, 274 differentially expressed immune-related genes (DEIRGs) were identified among six cell subpopulations in GSE112302 single-cell RNA sequencing (scRNA-seq) data of GC. Those DEIRGs were able to divide GC patients into three distinct subtypes with different overall survivals and tumor microenvironment. By univariate Cox and LASSO regression analyses, eight immune-related genes, including CTGF, CXCL3, CXCR4, NRP1, OAS1, SP1, STC1 and TAP1, were identified as GC prognostic signatures. Accordingly, a risk score model for predicting GC prognosis was constructed in TCGA-GC training cohort and validated in the external GSE66229 dataset. Moreover, a nomogram for predicting the survival of GC patients was also established based on independent prognostic factors (age, grade, cancer status and risk score) identified by univariate and multivariate Cox regression analyses. In addition, Gene Set Variation Analysis (GSVA) analysis indicated that the prognostic immune signatures may regulate GC via inflammation and cell proliferation related pathways, such as DNA replication, complement and coagulation cascades, focal adhesion and TGF-ß signaling pathway.

Shikonin attenuates rheumatoid arthritis by targeting SOCS1/JAK/STAT signaling pathway of fibroblast like synoviocytes.

BACKGROUND: Rheumatoid arthritis is a progressive and systemic autoimmune disease seriously compromises human health. Fibroblast like synoviocytes are the major effectors of proliferation and inflammation in rheumatoid arthritis synovial tissue. Shikonin has anti-inflammatory and immunomodulatory activities. But, its role on synovitis of rheumatoid arthritis is unknown. METHODS: The DBA/1 male mice were randomly divided into the following three groups (n = 6): (1) the normal control group of mice, (2) the CIA (collagen-induced arthritis) group in which mice suffered from arthritis induced by collagen, (3) the SKN (shikonin) group of mice which got arthritis and given intragastrically with shikonin 4 mg/kg per day continuously for 20 days,(4) the MTX (methotrexate) group of mice which got arthritis and orally administration with shikonin 0.5  mg/kg once two days continuously for 20 days. The therapeutic effect of shikonin on collagen induced arthritis mice was tested by arthritis incidence rate, arthritis score and inflammatory joint histopathology. The invasion, adhesion and migration of fibroblast like synoviocytes induced by tumor necrosis factor-α were applied to measure the anti-synovitis role of shikonin. The effect of shikonin on expression of interleukin-6, interleukin-1ß and tumor necrosis factor-α was measured by enzyme linked immunosorbent assay. The interaction between shikonin and suppressor of cytokine signaling 1 was verified by molecular docking. The signaling pathways activated by shikonin were measured by western blot. RESULTS: Shikonin decreased the arthritis score and arthritis incidence, and inhibited inflammation of inflamed joints in collagen induced arthritis mice. And shikonin reduced the number of vimentin+cells in collagen induced arthritis mice inflamed joints. Meanwhile, shikonin suppressed tumor necrosis factor-α-induced invasion, adhesion and migration of fibroblast like synoviocytes and reduced the expression of interleukin-6, interleukin-1ß and tumor necrosis factor-α. And we found that shikonin targeted suppressor of cytokine signaling 1. More interestingly, shikonin blocked the phosphorylation of Janus kinase 1/signal transducer andactivator of transcription 1/signal transducer andactivator of transcription 6 in synovial tissues and in fibroblast like synoviocytes. CONCLUSION: Shikonin represents a promising new anti-rheumatoid arthritis drug candidate that has anti-synovitis effect in collagen induced arthritis mice and inhibits tumor necrosis factor-α-induced fibroblast like synoviocytes by targeting suppressor of cytokine signaling 1/ Janus kinase/signal transducer andactivator of transcription signaling pathway. These findings demonstrate that shikonin has anti-synovitis effect and has great potential to be a new drug for the treatment of rheumatoid arthritis.

C-Reactive Protein Promotes the Activation of Fibroblast-Like Synoviocytes From Patients With Rheumatoid Arthritis.

Objective: To evaluate the biological effect and mechanisms of C-reactive protein (CRP) on the activation of fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). Study design: To understand if CRP is involved in RA, expression of CRP and its receptors CD32/64 was examined in synovial tissues from RA patients and normal controls. In vitro, the potential role and mechanisms of CRP in FLS proliferation and invasion, expression of pro-inflammatory cytokines, and activation of signaling pathways were investigated in both RA - FLS and a normal human fibroblast-like synoviocyte line (HFLS). Results: Compared to normal controls, synovial tissues from 21 RA patients exhibited highly activated CRP signaling, particularly by FLSs as identified by 65% of CRP-expressing cells being CRP+vimentin+ and CD32/64+vimentin+ cells. In vitro, FLSs from RA patients, but not HFLS, showed highly reactive to CRP by largely increasing proliferative and invasive activities and expressing pro-inflammatory cytokines and chemokines, including CCL2, CXCL8, IL-6, and MMP2/9. All these changes were blocked largely by a neutralizing antibody to CD32 and, to a less extent by the anti-CD64 antibody, revealing CD32 as a primary mechanism of CRP signaling during synovial inflammation. Further studies revealed that CRP also induced synovial inflammation differentially via CD32/CD64- NF-κB or p38 pathways as blockade of CRP-CD32-NF-κB signaling inhibited CXCL8, CCL2, IL-6, whereas CRP induced RA-FLS invasiveness through CD32-p38 and MMP9 expression via the CD64-p38-dependent mechanism. Conclusions: CRP signaling is highly activated in synovial FLSs from patients with RA. CRP can induce synovial inflammation via mechanisms associated with activation of CD32/64-p38 and NF-κB signaling.

Loss of Smad7 Promotes Inflammation in Rheumatoid Arthritis.

Objective: Smad7 is an inhibitory Smad and plays a protective role in many inflammatory diseases. However, the roles of Smad7 in rheumatoid arthritis (RA) remain unexplored, which were investigated in this study. Methods: The activation of TGF-ß/Smad signaling was examined in synovial tissues of patients with RA. The functional roles and mechanisms of Smad7 in RA were determined in a mouse model of collagen-induced arthritis (CIA) in Smad7 wild-type (WT) and knockout (KO) CD-1 mice, a strain resistant to autoimmune arthritis induction. Results: TGF-ß/Smad3 signaling was markedly activated in synovial tissues of patients with RA, which was associated with the loss of Smad7, and enhanced Th17 and Th1 immune response. The potential roles of Smad7 in RA were further investigated in a mouse model of CIA in Smad7 WT/KO CD-1 mice. As expected, Smad7-WT CD-1 mice did not develop CIA. Surprisingly, CD-1 mice with Smad7 deficiency developed severe arthritis including severe joint swelling, synovial hyperplasia, cartilage damage, massive infiltration of CD3+ T cells and F4/80+ macrophages, and upregulation of proinflammatory cytokines IL-1ß, TNFα, and MCP-1. Further studies revealed that enhanced arthritis in Smad7 KO CD-1 mice was associated with increased Th1, Th2 and, importantly, Th17 over the Treg immune response with overactive TGF-ß/Smad3 and proinflammatory IL-6 signaling in the joint tissues. Conclusions: Smad7 deficiency increases the susceptibility to autoimmune arthritis in CD-1 mice. Enhanced TGF-ß/Smad3-IL-6 signaling and Th17 immune response may be a mechanism through which disrupted Smad7 causes autoimmune arthritis in CD-1 mice.

Corrigendum to "Effect of Glycyrrhiza on the Diuretic Function of Euphorbia kansui: An Ascites Mouse Model".

[This corrects the article DOI: 10.1155/2016/7620817.].

Wu-tou decoction attenuates neuropathic pain via suppressing spinal astrocytic IL-1R1/TRAF6/JNK signaling.

Neuropathic pain (NP) caused by nerve injuries continues to be an intractable challenge due to inadequate therapeutic strategies. Recent study demonstrated glia-induced neuro-inflammation in the spinal cord, especially the activation of astrocytes, plays an essential role in the development of NP, which opens new avenues for NP treatment. In this study, we explored the anti-hyperalgesia properties of Wu-tou decoction (WTD) and showed that WTD potently attenuates mechanical allodynia and heat hyperalgesia in lumbar 5 (L5) spinal nerve ligation (SNL)-induced NP without noticeable side effect or affecting basal pain perception of mice. Mechanistically, initial targets screening tests indicated WTD's analgesic action may be centrally mediated within the spinal cord, which further verified by its inhibitory actions on glia-releasing factors of IL-1ß, CCL2 and CXCL1. Meanwhile, WTD significantly reduced spinal IL-1R1, TRAF6 expressions, p-JNK levels, and number of GFAP/IL-1R1, GFAP/TRAF6, GFAP/p-JNK positive astrocytes in the superficial lamina of spinal cord. Additionally, co-administration of IL-1Ra increased the anti-hyperalgesia effects of WTD and further decreased CCL2 and CXCL1 expressions, while no synergistic effects were detected when TRAF6 or JNK inhibitors were co-administrated with WTD. Thus, our data suggested that the effective inhibition of spinal astrocytic IL-1R1/TRAF6/JNK signaling (especially IL-1R1) contributes, at least in part, to WTD's anti-hyperalgesia action. It also indicates that WTD might be a promising candidate for the treatments of chronic pain, especially under NP-related neurological disorders.

Ethyl acetate fraction of Huogu formula inhibits adipogenic differentiation of bone marrow stromal cells via the BMP and Wnt signaling pathways.

Elevated adipogenesis of bone marrow stromal cells (BMSCs) is closely associated with non-traumatic osteonecrosis of femoral head (ONFH). Our previous studies have shown that Huogu (HG) formula was effective both in clinic experience and experimental ONFH. How HG impacts the differentiation of BMSCs and what is the underlying molecular mechanism remain largely unknown. Our results showed that ethyl acetate extract of HG (HGE) significantly decreased the adipocyte differentiation as determined by oil red staining, while slightly increased the ALP activity. Investigation of the molecular mechanism revealed that HGE could inhibit the mRNA and protein expression of peroxisome proliferators-activated receptor (PPAR)γ, lipoprotein lipase (LPL) and adipocyteprotein2 (AP2). Interestingly, the inhibition of adipogenic differentiation in BMSCs by HGE could be restored by DKK-1, an inhibitor of Wnts. However, Noggin (an inhibitor of BMPs) displayed an additive role with HGE in suppressing the expression of PPARγ, LPL, and AP2. Furthermore, the bone marrow fat formation, as well as the expression of Wnt3a and PPARγ, was effectively regulated by HGE in the steroid-induced ONFH rats. Our results demonstrated that HGE treatment significantly inhibited adipogenesis and slightly promoted osteogenesis of BMSCs through regulating the BMP and Wnt pathways. The findings shed lights on the molecular mechanism of HGE in the inhibition of adipogenesis and provide scientific rationale for its clinical application of HGE in the treatment of ONFH.

Forsythoside A exerts antipyretic effect on yeast-induced pyrexia mice via inhibiting transient receptor potential vanilloid 1 function.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel gated by noxious heat, playing major roles in thermoregulation. Forsythoside A (FT-A) is the most abundant phenylethanoid glycosides in Fructus Forsythiae, which has been prescribed as a medicinal herb for treating fever in China for a long history. However, how FT-A affects pyrexia and what is the underlying molecular mechanism remain largely unknown. Here we found that FT-A exerted apparent antipyretic effect through decreasing the levels of prostaglandin E2 (PGE2) and interleukin 8 (IL-8) in a dose-dependent fashion on the yeast induced pyrexia mice. Interestingly, FT-A significantly downregulated TRPV1 expression in the hypothalamus and dorsal root ganglion (DRG) of the yeast induced pyrexia mice. Moreover, FT-A inhibited IL-8 and PGE2 secretions, and calcium influx in the HEK 293T-TRPV1 cells after stimulated with capsaicin, the specific TRPV1 agonist. Further investigation of the molecular mechanisms revealed that FT-A treatment rapidly inhibited phosphorylation of extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 in both yeast induced pyrexia mice and HEK 293T-TRPV1 cells. These results suggest that FT-A may serve as a potential antipyretic agent and the therapeutic action of Fructus Forsythiae on pyretic related disease is, in part, due to the FT-A activities.

Effect of Glycyrrhiza on the Diuretic Function of Euphorbia kansui: An Ascites Mouse Model.

We investigated the therapeutic role of the herbal combination Euphorbia kansui (GS) and Glycyrrhiza (GC) in ascites during hepatocellular carcinoma (HCC). The AVPR2 and AQP2 expression in kidney tissues of ascites mice in different groups was determined by immunohistochemistry, Western blot, and real-time PCR analyses. When the dose of GS was less than 0.70 g/kg at a ratio of GC : GS not exceeding 0.4 : 1, the combination of GS and GC exhibited synergistic effects on HCC ascites and significantly elevated the expression levels of AVPR2 and AQP2 (all P < 0.05). On the contrary, when GS ≥ 0.93 g/kg and GC ≥ 1.03 g/kg with the GC-to-GS ratio exceeding 1.11 : 1, the combination of GS and GC displayed antagonistic effects on HCC ascites and dramatically reduced the expression levels of AVPR2 and AQP2 (all P < 0.05). Furthermore, the administration of herbal pair GS and GC at different ratios did not exacerbate the pathological changes in liver and kidney tissues of HCC ascites mice. The different combinations of GS and GC exerted synergistic or antagonistic effects on HCC ascites, partially by regulating the expression of AVPR2 and AQP2.

Aqueous Fraction of Huogu Formula Promotes Osteogenic Differentiation of Bone Marrow Stromal Cells Through the BMP and Wnt Signaling Pathways.

Our recent studies have shown that Huogu (HG) formula was effective both in clinic experience and in experimental osteonecrosis of the femoral head (ONFH). Given that defective of bone marrow stromal cells (MSCs) contribute to the development of osteonecrosis and MSCs show enormous potential in the treatment of ONFH, especially to aging people. How HG impacts the differentiation of MSCs and what is the underlying cellular and molecular mechanism remains largely unknown. Here, we found that an aqueous fraction of HG (HGA) significantly increased the alkaline phosphatase (ALP) activity, mineralized nodules, and migration of MSCs in a dose-dependent manner. Meanwhile, HGA could enhance the mRNA and protein expression of Runt-related transcription factor 2 (Runx2), Alp, Bmp2, osteocalcin (Ocn), and Osterix (Osx). Further investigation of the molecular mechanisms revealed that HGA treatment obviously increased expression, secretion, and activation of bone morphogenetic protein (BMP) 2 and ß-catenin, two key regulators of the BMP or Wnt signaling pathway. Furthermore, osteogenic differentiation of MSCs could be blocked by using pharmacological inhibitors for these signaling pathways such as Noggin and Dkk-1. Besides, HGA could inhibit adipogenic differentiation of MSCs. Our study reveals that HGA promotes the osteogenesis of MSCs via the BMP and Wnt signaling pathways. Our findings provide mechanistic insights into the role of HG in treating ONFH.

Tetramethylpyrazine Promotes Migration of Neural Precursor Cells via Activating the Phosphatidylinositol 3-Kinase Pathway.

Stem cell therapy may provide a novel therapeutic method for the replacement and regeneration of damaged neural cells in the central nervous system. However, insufficient stem cell migrating into the injured regions limits its applications. Although tetramethylpyrazine (TMP) originally isolated from Ligusticum walliichi (Chuanxiong) has been widely used to treat ischemic stroke in the clinic for many years because of its role in neuroprotection, how TMP impacts the migration of neural progenitor/precursor cells (NPCs) and what is the underlying cellular and molecular mechanism remain largely unknown. Here, we found that TMP promoted NPC migration through increasing the expression and secretion of stromal cell-derived factor 1 (SDF-1), a chemokine that has been well demonstrated to direct NPC cell trafficking, in a dose-dependent fashion as analyzed by using different methods. The role of TMP in NPC migration could be inhibited by AMD 3100, a chemokine (C-X-C motif) receptor 4 (CXCR4) antagonist. Further investigation of the molecular mechanisms revealed that TMP treatment rapidly activated phosphatidylinositol 3-kinase (PI3K)/Akt, protein kinase C (PKC), and extracellular signal-regulated kinase (ERK), but not Pyk2, in NPCs. NPC migration could be blocked by using pharmacological inhibitors for these signaling pathways such as LY294002 (a PI3K inhibitor), Myr-ψPKC (a PKC inhibitor), and an ERK1/2 inhibitor. Furthermore, TMP enhanced NPC migration toward the ischemic region in the MCAO rat model. Our findings provide mechanistic insights into the role of TMP in treating the neuropathological diseases, which suggest that TMP may be used as a potent drug for improving NPC migration in stem cell-based therapy.