Xiaochen Guo

Fibroblast growth factor 21 (FGF21) inhibits macrophage-mediated inflammation by activating Nrf2 and suppressing the NF-κB signaling pathway

Our previous report has shown that FGF21 has anti-inflammatory properties in a collagen-induced arthritis (CIA) model. In this study, the underlying molecular mechanisms of action were also investigated using RAW 264.7 cells, a murine monocyte-macrophage. RAW 264.7 cells were pre-incubated with various concentrations (2000, 500, 100ng/ml) of FGF21 and stimulated with LPS to induce oxidative stress and inflammation. The result of flow cytometry showed that β-Klotho, FGF21 specific receptor, was expressed in murine splenic macrophages and RAW 264.7. In vitro, FGF21 reduced the expression of TNF-α, IL-1β, IL-6 and IFN-γ and increased the level of IL-10 in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. FGF21 also suppressed profound elevation of ROS production and oxidative stress, as evidenced by an increase of the MDA level and depletion of the intracellular GSH level, and restored the activities of antioxidant enzymes SOD and GSH-Px in LPS-stimulated RAW 264.7 macrophages. Moreover, FGF21 inhibited LPS-induced nuclear factor-κB (NF-κB) activation, including degradation of I-κB and nuclear translocation of p65. In addition, the result of Western blot and real-time PCR showed that FGF21 induced heme oxygenase-1 (HO-1) expression and increased the nuclear transcription factor-E2-related factor 2 (Nrf2) levels in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. In conclusion, the results suggest that macrophages are the targets for the anti-inflammatory effects of FGF21, and FGF21 exerted an anti-inflammatory effect mainly via enhancing Nrf2-mediated anti-oxidant capacity and suppressing NF-κB signaling pathway.

Fibroblast growth factor 21 ameliorates high glucose-induced fibrogenesis in mesangial cells through inhibiting STAT5 signaling pathway

Fibroblast growth factor 21 (FGF21) is a member of the FGF family and acts as a potent regulator of glucose and lipid homeostasis, but its effect on renal fibrosis and the underlying mechanisms are totally unknown. The purpose of this study was designed to investigate whether FGF21 has effect on high glucose-induced fibrogenesis in human mesangial cells (HMCs) and the underlying mechanism. High glucose is well known to stimulate the expression of extracelluar matrix (ECM) in human mesangial cells. In humans, overexpression and deposition of ECM lead to renal fibrosis. Thus, in this study, HMCs were incubated in high glucose with or without various concentrations of FGF21. Results demonstrated that the expression of FN, Col, TGF β1 and α-SMA were significantly up-regulated in HMCs. Whereas, treatment with FGF21 down-regulated the expression of FN, Col, TGF β1 and α-SMA. In addition, growth factors are considered to be an important driving force for the pathogenesis of renal fibrosis. Significantly increased PDGF, VEGF and CTGF expression were found in HMCs induced by high glucose. FGF21 treatments significantly decreased these growth factors expression by down-regulating the phosphorylation level of STAT5. Here, we reported for the first time that FGF21 decreases ECM expression by inhibiting STAT5 signal pathway and consequently decreasing the expression of PDGF, VEGF and CTGF.

Improving hyperglycemic effect of FGF-21 is associated with alleviating inflammatory state in diabetes

&NA; Type 2 diabetes mellitus (T2DM) is accompanied by abnormal glucose metabolism and low‐grade chronic inflammation. Fibroblast growth factor 21 (FGF‐21) is a novel metabolic regulator and can function as an endocrine hormone to regulate glucose and lipid metabolism. Recently, FGF‐21 was found to have anti‐inflammatory effect, to our knowledge, the effect of FGF‐21 on inflammatory state in diabetes has not been elucidated. Here, we use db/db mice as a Type 2 diabetes model to determine whether FGF‐21 alleviates inflammatory state while improves hyperglycemia. Our results demonstrated that FGF‐21 not only showed potent long lasting hypoglycemic effect, but also demonstrated strong anti‐inflammatory effect in the serum and white adipose tissue. Besides, in vitro experiments, insulin resistance (IR) was induced in 3T3‐L1 adipocytes by treating with TNF‐&agr;. Our results showed that TNF‐&agr; impaired glucose metabolism of 3T3‐L1 adipocytes but FGF‐21 repressed gene expression of inflammatory factors caused by IR and consequently improved the glucose metabolism in 3T3‐L1 adipocytes. Furthermore, FGF‐21 ameliorated glucose uptake of TNF‐&agr;‐induced IR in 3T3‐L1 adipocytes by inhibiting NF‐&kgr;B signaling pathway.

A recombinant IgG-like bispecific antibody acting as interleukin-1β and interleukin-17A inhibitor exhibits a promising efficacy for rheumatoid arthritis

Recently, targeting inflammatory cytokines in the pathogenic process of rheumatoid arthritis is now performed as a feasible biological method in therapy. However, treatments against single cytokine are often difficult to achieve the ideal therapeutic effect. Multi-target drugs permit more effective suppression of inflammation. In this study, we constructed an IgG-like bispecific antibody targeting IL-1β and IL-17A and expressed it in mammalian cells. The therapeutic efficacy was studied in CIA (collagen-induced arthritis) mice, which were administrated with either FL-BsAb1/17 (IgG-like bispecific antibody targeting IL-1β and IL-17A) or monovalent IL-1β Mab or IL-17A Mab (anti-IL-1β/IL-17A monoclonal antibody). We noticed that FL-BsAb1/17 had better effect on alleviating clinical symptom by significantly lowering arthritis score and relieving histological lesion on aspect of less damnification in synovial hyperplasia and cartilage destruction than monovalent Mab alone. In addition, FL-BsAb1/17 was more potent in inhibiting IL-1β, IL-17A, IL-6, TNF-α and anti-CCP antibody in the serum and in down-regulating the expression of IL-1β, IL-17A, IL-6, TNF-α, MMP-3 and RANKL in the spleen, compared to monovalent Mab alone. Further, the anti-inflammatory effect of FL-BsAb1/17 was demonstrated by significantly depressing Th17 cells expansion through decreasing phosphorylated STAT3 in the spleen of the CIA mice. FLS (fibroblast-like synoviocytes) from RA patients were used to examine the therapeutic efficacy of FL-BsAb1/17 in human pathological tissue. FL-BsAb1/17 could significantly decrease the production of IL-6 induced by IL-1β and/or IL-17A in FLS. In conclusion, FL-BsAb1/17 has the possibility to be a promising therapeutic agent for RA.

Newcastle disease virus chimeras expressing the Hemagglutinin- Neuraminidase protein of mesogenic strain exhibits an enhanced anti-hepatoma efficacy.

Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, many researchers have reported that lentogenic NDV is a safe and effective agent for human cancer therapy. It had been demonstrated that the amino acid sequence of the fusion protein cleavage site is a major factor in the pathogenicity and anti-tumor efficacy of rNDV. However, the role of Hemagglutinin-Neuraminidase (HN) gene that contributes to virulence and anti-tumor efficacy remains undefined. To assess the role of HN gene in virus pathogenicity and anti-tumor efficacy, a reverse genetic system was developed using the lentogenic NDV Clone30 strain to provide backbone for gene exchange. Chimeric virus (rClone30-Anh(HN)) created by exchange of the HN gene of lentogenic strain Clone30 with HN gene of mesogenic strain produce no significant changes in virus pathogenicity as assessed by conducting the mean death time (MDT) and intracerebral pathogenicity index (ICPI) assays. In vitro, infection with chimeras could induce the formation of syncytium relative significantly in HepG2 cells. Furthermore, chimeras was shown to induce the cell apoptosis via MTT and Annexin V-PI assays, reduce mitochondrial membrane potential and increase the mRNA transcription level of caspase 3. In vivo, ICR mice carrying tumor of hepatoma H22 cells were treated via intratumoral injection of chimeric virus. The treatment of chimera shows an obvious suppression in tumor volume. These results suggest that it could be an ideal approach to enhance the antitumor ability of Newcastle disease virus and highlighted the potential therapeutic application of rClone30-Anh(HN) as a viral vector to deliver foreign genes for treatment of cancers.

Screening scFv antibodies against infectious bursal disease virus by co-expression of antigen and antibody in the bacteria display system.

We have previously reported an antigen and antibody co-expression (AAC) technology to demonstrate the interaction between a known antigen and antibody. To validate the co-expression system for screening antibody libraries, a single chain fragment variable(scFv)antibody library was constructed from chickens immunized with the VP2 protein of infectious bursal disease virus (IBDV). The genes of both VP2 and scFv antibodies were inserted into the pBFD-Ab-Ag vector. The co-expression library was subjected to three rounds of screening by flow cytometry (FCM) using a polyclonal antibody against VP2 through a bacteria display system. We achieved enrichment of scFv specific for IBDV. 110 individual clones were initially selected and sequenced. Twenty clones were selected based on fluorescence intensity by FCM. The scFv antibodies were expressed by pET-27b in E.coli and purified. The specificity and affinity of the selected scFv antibodies were confirmed by western blotting assay and ELISA analysis. Whats more, the neutralizing capacity was measured with IBDV-B87(100 TCID50) in vitro. Four scFvs (clone 8(1), Y8, L10 and L7) showed significant neutralizing capacity. Two of the four scFvs (clone 8(1) and Y8) demonstrated a higher neutralizing activity to IBDV-B87 and the titers were 16,384 and 8,192, respectively. The two scFvs had higher neutralizing capacity than those obtained in previous studies. We demonstrated that the AAC technology could be applied to screen antibody libraries without baiting antigen to make antibody screening process easier and obtain scFvs with higher neutralizing capacity.

Selection of affinity-improved neutralizing human scFv against HBV PreS1 from CDR3 VH/VL mutant library.

A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin.

A novel fusion protein attenuates collagen–induced arthritis by targeting interleukin 17A and tumor necrosis factor α

Graphical abstract Figure. No caption available. ABSTRACT This study was undertaken to generate a novel dual targeting fusion protein (DTF), targeting tumor necrosis factor &agr; (TNF‐&agr;) and interleukin 17A (IL‐17A), and determine its anti‐arthritis properties in vitro and in vivo. DTF consisted of an anti‐IL‐17A single chain variable fragment, a soluble TNF receptor 1, and an Fc fragment. Both clinical and histopathological evaluations suggest that DTF and etanercept can ameliorate collagen induced arthritis. However, the arthritis severity score of DTF‐treated mice was lower than that of etanercept‐treated mice. In addition, DTF was more potent than etanercept in decreasing the ratio of RANKL/OPG in the serum and rebalancing the population ratio of Treg/Th17 cells in the spleens. In vitro, IL‐17A and TNF‐&agr; had synergistic effects in inducing the expression of inflammatory cytokines in fibroblast‐like synoviocyte from RA patients (RA‐FLS), human leukemia (THP‐1), and rheumatoid synovial fibroblast (MH7A). IL‐17A and TNF‐&agr; also had synergistic effects in inducing proliferation and migration of MH7A cells. However, we observed that DTF was more efficient than etanercept in suppressing these synergistic effects. Our results demonstrate that DTF is highly efficient in the treatment of arthritis and has the potential to overcome the limited therapeutic responses obtained with single cytokine neutralization.

Fibroblast growth factor 21 regulates foam cells formation and inflammatory response in Ox-LDL-induced THP-1 macrophages

Macrophages are paramount to the initiation and procession of atherosclerosis, thus targeting macrophages in the progress of atherosclerosis is indispensable. Therefore, we perform in vitro experiments to investigate the effects of FGF-21 on macrophages in the progress of atherosclerosis. First, we use phorbol-12-myristate-13-acetate (PMA), a phorbol ester, to induce THP-1 cells into macrophages as macrophages model. After that we use Ox-LDL to induce macrophages into foam cells and simultaneously administrate with FGF-21 or not to determine whether FGF-21 has effects on foam cells formation and related inflammatory response. Wound healing results show that FGF-21 can inhibit macrophage migration. Oil Red-O stain, immunofluorescence and flow cytometer results show that FGF-21 can repress cholesterol accumulation in macrophages thereby inhibit foam cells formation and these effects can be abolished by FGFR inhibitor. Moreover, real-time PCR results showed that FGF-21 significantly reduces expression of inflammatory factors including IL-1α, IL-6 and TNF-α and this effect can be abolished by FGFR inhibitor. Furthermore, to determine the mechanism of FGF-21 regulates inflammatory response in Ox-LDL-induced THP-1 macrophages, western blotting results show that after treatment of Ox-LDL in macrophages, NF-κB signaling pathway is activated but FGF-21 can significantly inhibit this pathway. In addition, FGF-21 also regulates some regulators of lipid metabolism after treatment of Ox-LDL in macrophages. Above all, our findings demonstrate that FGF-21 can regulate foam cells formation, macrophage migration, inflammatory response and lipid metabolism in Ox-LDL-induced THP-1 macrophages.

Fibroblast growth factor 21 regulates glucose metabolism in part by reducing renal glucose reabsorption

Although previous studies have shown the potential of FGF21 to regulate blood glucose in animal and humans, the precise mechanisms of the action have not been well explored. The kidney plays a crucial role for glucose homeostasis. The purpose of this study is to explore the effect of FGF21 on renal glucose reabsorption. Administration of type 2 and type 1 diabetic mice with FGF21 reduced the transport maximum of glucose in the kidney and enhanced urinary glucose excretion in a dose-dependent manner. The inhibition of glucose reabsorption results showed little change in diabetic mice treated with Insulin. In physiological state, both FGF21 and insulin had no effect on glucose reabsorption and urinary glucose excretion. Next, we examined the expression of SGLT2 in the kidney, which is an important molecule for renal glucose reabsorption. SGLT2 was highly expressed in the kidneys of diabetic mice. Administration of FGF21 reduced SGLT2 expression in the kidney of diabetic mice. In contrast, the expression of SGLT2 had little change in diabetic mice treated with Insulin. FGF21 and Insulin did not promote SGLT2 expression in physiological state. To explore the mechanism which drives these changes, we detected the expression of PPARδ in mice and HK-2 cells, which plays a major role in regulating SGLT2 expression. Treatment with FGF21 promoted PPARδ expression in diabetic mice, whereas Insulin had no effect on PPARδ expression. At dose of 2 mg/kg FGF21 treatment promoted PPARδ expression in physiological state, whereas at dose of 1 mg/kg FGF21 did not. In HK-2 cells, treatment with FGF21 enhanced PPARδ expression, whereas Insulin treatment had no effect on PPARδ expression. Importantly, the expression of SGLT2 and PPARδ showed little change in HK-2 cells when β-klotho was knocked down. In conclusion, we discovered for the first time that FGF21 ameliorates hyperglycemia in part via reducing renal glucose reabsorption through PPARδ mediated SGLT2 pathway.