Shuai Jiang

Effects of multiple genetic loci on the pathogenesis from serum urate to gout

Gout is a common arthritis resulting from increased serum urate, and many loci have been identified that are associated with serum urate and gout. However, their influence on the progression from elevated serum urate levels to gout is unclear. This study aims to explore systematically the effects of genetic variants on the pathogenesis in approximately 5,000 Chinese individuals. Six genes (PDZK1, GCKR, TRIM46, HNF4G, SLC17A1, LRRC16A) were determined to be associated with serum urate (PFDR < 0.05) in the Chinese population for the first time. ABCG2 and a novel gene, SLC17A4, contributed to the development of gout from hyperuricemia (OR = 1.56, PFDR = 3.68E-09; OR = 1.27, PFDR = 0.013, respectively). Also, HNF4G is a novel gene associated with susceptibility to gout (OR = 1.28, PFDR = 1.08E-03). In addition, A1CF and TRIM46 were identified as associated with gout in the Chinese population for the first time (PFDR < 0.05). The present study systematically determined genetic effects on the progression from elevated serum urate to gout and suggests that urate-associated genes functioning as urate transporters may play a specific role in the pathogenesis of gout. Furthermore, two novel gout-associated genes (HNF4G and SLC17A4) were identified.

Sirtuin1 Protects against Systemic Sclerosis–related Pulmonary Fibrosis by Decreasing Proinflammatory and Profibrotic Processes

&NA; Pulmonary fibrosis is the leading cause of death in systemic sclerosis (SSc). Sirtuin1 (SIRT1) is a deacetylase with known antiinflammatory and antifibrotic activity in the liver, kidney, and skin. The role of SIRT1 in SSc‐related pulmonary fibrosis is unknown. In the present work, we determined that the expression of SIRT1 in peripheral blood mononuclear cells of patients with SSc with pulmonary fibrosis is lower than that in patients with SSc without pulmonary fibrosis. In in vivo studies of bleomycin‐induced lung fibrosis in mice, SIRT1 activation with resveratrol reduced collagen production when it was administered either prophylactically during the inflammatory stage or after the development of fibrosis. Furthermore, SIRT1 activation or overexpression inhibited tumor necrosis factor‐&agr;‐induced inflammatory responses in vitro in human fetal lung fibroblasts, depletion of SIRT1 in fibroblasts enhanced inflammation, and these effects were related to changes in the acetylation of NF‐&kgr;B. In addition, SIRT1 activation or exogenous overexpression inhibited collagen production in vitro, and these manipulations also inhibited fibrosis via inactivation of transforming growth factor‐&bgr;/mothers against decapentaplegic homolog and mammalian target of rapamycin signaling. Taken together, our results show that a loss of SIRT1 may participate in the pathogenesis of SSc‐related pulmonary fibrosis, and that SIRT1 activation is an effective treatment for both the early (inflammatory) and late (fibrotic) stages of pulmonary fibrosis. Thus, SIRT1 may be a promising therapeutic target in the management of SSc‐related pulmonary fibrosis.

Treatment effects of the traditional Chinese medicine Shenks in bleomycin-induced lung fibrosis through regulation of TGF-beta/Smad3 signaling and oxidative stress

Pulmonary fibrosis is a kind of devastating interstitial lung disease due to the limited therapeutic strategies. Traditional Chinese medicine (TCM) practices have put forth Shenks as a promising treatment approach. Here, we performed in vivo study and in vitro study to delineate the anti-fibrotic mechanisms behind Shenks treatment for pulmonary fibrosis. We found that regardless of the prophylactic or therapeutic treatment, Shenks was able to attenuate BLM-induced-fibrosis in mice, down regulate extracellular matrix genes expression, and reduce collagen production. The aberrantly high Smad3 phosphorylation levels and SBE activity in TGF-β-induced fibroblasts were dramatically decreased as a result of Shenks treatment. At the same time, Shenks was able to increase the expression of antioxidant-related genes, including Gclc and Ec-sod, while reduce the transcription levels of oxidative-related genes, such as Rac1 and Nox4 demonstrated by both in vivo and in vitro studies. Further investigations found that Shenks could decrease the oxidative productions of protein (3-nitrotyrosine) and lipid (malondialdehyde) and increase GSH content both in bleomycin treated mouse lungs and TGF-β stimulated fibroblasts, as well as inhibit the production of ROS stimulated by TGF-β to fight against oxidative stress. Overall, Shenks inhibited fibrosis by blocking TGF-β pathway and modulating the oxidant/antioxidant balance.

Increased expression of latent TGF- β -binding protein 4 affects the fibrotic process in scleroderma by TGF- β /SMAD signaling

Scleroderma is a fibrosis-related disorder characterized by cutaneous and internal organ fibrosis, and excessive collagen deposition in extracellular matrix (ECM) is a major cause of fibrosis. Transforming growth factor-β (TGF-β)/SMAD signaling has a central role in the pathogenesis of fibrosis by inducing abnormal collagen accumulation in ECM, and latent TGF-β-binding protein 4 (LTBP-4) affects the secretion of latent TGF-β to ECM. A previous study indicated that bleomycin (BLM) treatment increased LTBP-4 expression in lung fibroblasts of Thy-1 knockout mice with lung fibrosis, and LTBP-4 further promoted TGF-β bioavailability as well as SMAD3 phosphorylation. However, the expression and function of LTBP-4 in human scleroderma remain unclear. We aimed to investigate the potential role of LTBP-4 in scleroderma through clinical, in vivo and in vitro studies. LTBP-4 and TGF-β expressions were significantly upregulated in systemic scleroderma (SSc) patients’ plasma compared with normal controls (LTBP-4, 1,215±100.2 vs 542.8±41.7 ng/ml, P<0.0001; TGF-β, 1.5±0.2 vs 0.7±0.1 ng/ml, P=0.0031), while no significant difference was found between localized scleroderma (LSc) and normal controls. The plasma concentrations of LTBP-4 and TGF-β were even higher in SSc patients with lung fibrosis (LTBP-4, 1462± 137.3 vs 892.8±113.4 ng/ml, P=0.0037; TGF-β, 2.0±0.4 vs 0.9±0.2 ng/ml, P=0.0212) and esophagus involvement (1390±134.4 vs 940.7±127.0 ng/ml, P=0.0269; TGF-β, 1.9±0.3 vs 0.9±0.2 ng/ml, P=0.0426). The area under receiver operating characteristics (ROC) curve of LTBP-4 was 0.86. Immunohistochemistry measurement also demonstrated a higher LTBP-4 expression in sclerotic skin tissue of LSc and SSc compared with normal controls. More positive fibroblasts were also found in BLM-induced scleroderma mouse model than the saline-treated group. In in vitro studies, knockdown of LTBP-4 in SSc skin fibroblasts prominently reduced downstream COL1A1, COL1A2, and COL3A1 mRNA level by 84%, 82%, and 43%, respectively, and other fibrosis-related genes’ expression were also decreased. Furthermore, extracellular TGF-β level and the SMAD2/3 phosphorylation were inhibited through LTBP-4 knockdown treatment, suggesting that the knockdown of LTBP-4 reduced the collagen expression through TGF-β/SMAD signaling pathway. Taken together, these data suggest that LTBP-4 affects fibrotic process in scleroderma, and the high expression of LTBP-4 in SSc plasma may serve as a clinical biomarker in diagnosing this disease. In addition, this study also lays the theoretical foundation for targeting LTBP-4 as treatment of scleroderma.

Copy number variants of ABCF1, IL17REL, and FCGR3A are associated with the risk of gout

Gout is usually characterized by recurrent flares of acute inflammatory arthritis and the crystallization of urate in tissues and joints (Dong et al., 2017). The formation of monosodium urate crystals (MSU) is due to the elevated uric acid levels in the blood (Yang et al., 2016). But only 10% of individuals with hyperuricemia develop gout, suggesting that certain people are at a higher risk of developing MSU crystals (Merriman and Dalbeth, 2011) or initiating of the acute inflammatory response than others. This phenomenon is likely explained by the complex pathogenic process of inflammation and innate immunity in the development of gout since theMSU crystal-mediated inflammatory pathways have been reported to associate with many immunological processes (Behrens et al., 2008). Theabove results suggest that inflammationand immunity-related factors can regulate the development of gout, particularly the progression from hyperuricemia to gout. Recent genome-wide association studies (GWAS) identified some single nucleotide polymorphisms (SNPs) that were associated with the risk of gout (Kottgen et al., 2013). However, the common SNPs only explained part of the heritability of gout, and copy number variants (CNVs), an important source of genetic diversity in humans, have been shown to play a critical role in the genetic susceptibility to multiple rheumatic diseases (Zhang et al., 2009). Therefore, CNVs might be a new source for the genetic variants associated with the pathogenesis of gout. However, CNV studies on gout have not been reported, making it necessary to explore the pathogenesis of gout through the copy number variants in the inflammationand immunity-related genes. In this study, we aimed to identify the gout-associated CNVs in a Chinese population using a three-step approach. The entire analysis strategy, from genome-wide CNV discovery to gout-associated CNV identification to CNV validation, was illustrated in Fig. S1. Using this strategy, we provided the first evidence that CNVs could influence the pathogenesis of gout, and identified three novel genes, ABCF1, IL17REL and FCGR3A, which are associated with the risk of gout. Based on a genome-wide analysis of CNVs, a total of 1,901 CNVs were identified in 22 patients with inflammationand immunity-related rheumatic disorders (inclunding gout, ankylosing spondylitis, systemic lupus erythematosus, and systemic sclerosis) compared to the controls by using Agilent SurePrint G3 Human 1 × 1 M comparative genomic hybridization (CGH) microarray. After they were filtered according to their frequency (>5% and ≤50%), genome location (in gene region), gene function (related with inflammation and immunity), reliability (at least five consecutive probes) and previous studies for these CNVs, 48 gene regions with CNVs were selected (Table S1). To identify candidate gout-associated CNVs, the above pre-selected CNVs were further tested in 46 gout patients and 47 healthy Chinese individuals using the NimbleGen CGH Arrays (12 × 270 K), which is a high-density CGH assay, designed and made by Roche. Five CNVs appeared in more than 5% of the subjects, and located at a chromosome region that contained exons for inflammationand immunity-related genes (ABCF1, IL17REL, FCGR3A, DPCR1, and DEFA10P), and were identified to be candidate CNVs associated with gout. To validate the significance of the CNVs, the above five candidate CNVs (Table S2) were tested in an additional 1,274 Chinese individuals (576 gout patients and 698 control subjects, Tables S3 and S4) using AccuCopy (Du et al., 2012). The distributions of three CNVs in ABCF1, IL17REL, and FCGR3A were significantly different between the gout patients and the controls according to Fisher’s exact test, with P values of 0.018, 0.021 and 0.022, respectively (Tables S5 and S6). After correcting for multiple comparisons, the distributions of these CNVs were still significantly different (all P = 0.037) (Table S5). In addition, to avoid the heterogeneity of gender and age, deviance analysis for the logistic regression model adjusted for gender and age also showed that the CNVs in IL17REL and FCGR3A were significantly different between gout patients and control subjects (P = 0.002 and 0.038, respectively). The other two CNVs in the DPCR1 and DEFA10P genes were not associated with the risk of gout in the Chinese population (P = 0.328 and 0.221, respectively). The ABCF1 gene encodes a superfamily member of the ATP-binding cassette (ABC) transporters. The ABC

Sirt1 ameliorates systemic sclerosis by targeting the mTOR pathway.

BACKGROUND Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by inflammation and fibrosis. Our previous research has indicated that Sirtuin1 (Sirt1) plays a role in the regulation of TNF-α-induced inflammation; however, whether Sirt1 may inhibit the progress of SSc by blocking inflammation remains unknown. OBJECTIVE We aimed to investigate the function of Sirt1 in SSc. METHODS The function and its mechanism of Sirt1 were evaluated in fibroblasts or scleroderma mice. The expression of Sirt1 and cytokines was analyzed using real-time PCR, western blot, ELISA and immunohistochemistry. RESULTS We determined that fibroblasts of SSc patients were activated to exhibit inflammation. Sirt1, activated by resveratrol (Res), ameliorated cutaneous inflammation and fibrosis in bleomycin (BLM)-induced scleroderma mice. An improvement in mammalian target of rapamycin (mTOR) was identified in the fibroblasts of SSc patients and the skin lesions of BLM mice. Rapamycin, an mTOR specific inhibitor, substantially inhibited the induced inflammation and fibrosis. The enhancement of mTOR expression in the skin lesions of the BLM-treated mice was significantly inhibited by Sirt1 activation. However, in both the BLM-treated cells and mice, Res exerted an inhibitory function on the expression of inflammatory factors, and collagen was diminished following mTOR knockdown. These findings suggest that Res may inhibit inflammation and fibrosis via mTOR. CONCLUSION The modulation of Sirt1 activity may represent a potential therapeutic method for SSc. The mechanism may involve the inhibition of mTOR phosphorylation, whereas mTOR activity was shown to be a pathogenic culprit of SSc.

Evaluation of the antifibrotic potency by knocking down SPARC, CCR2 and SMAD3

The genes of SPARC, CCR2, and SMAD3 are implicated in orchestrating inflammation and fibrosis in scleroderma and other fibrotic disorders. Aim of the studies was to examine synergistic effect of inhibition of these genes in treating fibrosis. The peptide nanoparticles were used to deliver the siRNAs in bleomycin-induced fibrotic mice. Triple combination of siRNAs targeting on Sparc, Ccr2 and Smad3 achieved favorable anti-inflammatory and anti-fibrotic effects. Inhibition of inflammation was evidenced by reduced inflammatory cells and proinflammatory cytokines in the BALF and/or the tissues. Activation of fibroblasts was suppressed in mouse tissues in which α-Sma and collagens were significantly reduced. Aberrant expression of the genes in fibroblasts, monocytes/macrophage, endothelial and epithelial cells were reinstalled after the treatment. In addition, transcriptome profiles indicated that some bleomycin-induced alterations of multiple biological pathways were recovered to varying degrees by the treatment. The results indicated that the triple combination of siRNAs systemically reinstated multiple biopathways, probably through controlling on different cell types including fibroblasts, monocytes/macrophages, endothelial cells and others. The multi-target-combined therapeutic approach examined herein may represent a novel and effective therapy for fibrosis.

PKD2 influence uric acid levels and gout risk by interacting with ABCG2

Background Uric acid is the final product of purine metabolism and elevated serum urate levels can cause gout. Conflicting results were reported for the effect of PKD2 on serum urate levels and gout risk. Therefore, our study attempted to state the important role of PKD2 in influencing the pathogenesis of gout. Method SNPs in PKD2 (rs2725215 and rs2728121) and ABCG2 (rs2231137 and rs1481012) were tested in approximately 5,000 Chinese individuals. Results Two epistatic interactions between loci in PKD2 (rs2728121) and ABCG2 (rs1481012 and rs2231137) showed distinct contributions to uric acid levels with P int values of 0.018 and 0.004, respectively, and the associations varies by gender and BMI. The SNP pair of rs2728121 and rs1481012 justly played roles in uric acid in females (P int = 0.006), while the other pair did in males (P int = 0.017). Regarding BMI, the former SNP pair merely contributed in overweigh subjects (P int = 0.022) and the latter one did in both normal and overweigh individuals (P int = 0.013 and 0.047, respectively). Furthermore, the latter SNP pair was also associated with gout pathology (P int = 0.001), especially in males (P int = 0.001). Finally, functional analysis showed potential epistatic interactions in those genes region and PKD2 mRNA expression had a positive correlation with ABCG2’s (r = 0.743, P = 5.83e-06). Conclusion Our study for the first time identified that epistatic interactions between PKD2 and ABCG2 influenced serum urate concentrations and gout risk, and PKD2 might affect the pathogenesis from elevated serum urate to hyperuricemia to gout by modifying ABCG2.

Genetic variants in two pathways influence serum urate levels and gout risk: a systematic pathway analysis

The aims of this study were to identify candidate pathways associated with serum urate and to explore the genetic effect of those pathways on the risk of gout. Pathway analysis of the loci identified in genome-wide association studies (GWASs) showed that the ion transmembrane transporter activity pathway (GO: 0015075) and the secondary active transmembrane transporter activity pathway (GO: 0015291) were both associated with serum urate concentrations, with PFDR values of 0.004 and 0.007, respectively. In a Chinese population of 4,332 individuals, the two pathways were also found to be associated with serum urate (PFDR = 1.88E-05 and 3.44E-04, separately). In addition, these two pathways were further associated with the pathogenesis of gout (PFDR = 1.08E-08 and 2.66E-03, respectively) in the Chinese population and a novel gout-associated gene, SLC17A2, was identified (OR = 0.83, PFDR = 0.017). The mRNA expression of candidate genes also showed significant differences among different groups at pathway level. The present study identified two transmembrane transporter activity pathways (GO: 0015075 and GO: 0015291) were associations with serum urate concentrations and the risk of gout. SLC17A2 was identified as a novel gene that influenced the risk of gout.

Salvianolic acid B attenuates experimental pulmonary inflammation by protecting endothelial cells against oxidative stress injury

ABSTRACT Endothelial cell injury and subsequent inflammation play pivotal roles in the pathogenesis of pulmonary fibrosis, a progressive and fatal disorder. We found previously that salvianolic acid B (SAB) attenuated experimental pulmonary fibrosis. Pulmonary fibrosis is driven by inflammation, but the anti‐inflammatory role and mechanism of SAB on the treatment of pulmonary fibrosis is still unknown. Here, our in vivo studies showed that SAB had a strong anti‐inflammatory effect on bleomycin‐instilled mice by inhibiting inflammatory cell infiltration and inflammatory cytokine production. Moreover, SAB protected endothelial cells against oxidative stress injury and inhibited endothelial cell apoptosis in bleomycin‐treated mice. The in vitro studies also showed that SAB decreased the H2O2‐induced overproduction of reactive oxygen species to protect EA.hy926 endothelial cells from oxidative damage, and further inhibited H2O2‐induced permeability and overexpression of pro‐inflammatory molecules. The next studies revealed that SAB inhibited the H2O2‐induced cell apoptosis and attenuated the decrease of tight junction‐related gene expression, resulting in a decrease of the endothelial permeability in injured endothelial cells. Furthermore, Western blot analysis suggested that SAB decreased endothelial cell permeability and expression of pro‐inflammatory cytokines by inhibiting MAPK and NF‐&kgr;B signaling pathways. Taken together, these data indicate that SAB exerted anti‐inflammatory roles in pulmonary fibrosis by protection of the endothelial cells against oxidative stress injury, mediated by inhibition of endothelial permeability and expression of pro‐inflammatory cytokine via the MAPK and NF‐&kgr;B signaling pathways. Graphical abstract Figure. No caption available.