Sparsentan ameliorates glomerular hypercellularity and inflammatory-gene networks induced by IgA1-IgG immune complexes in a mouse model of IgA nephropathy.

IgA nephropathy (IgAN) is characterized by glomerular deposition of immune complexes (ICs) consisting of IgA1 with O-glycans deficient in galactose (Gd-IgA1) and Gd-IgA1-specific IgG autoantibodies. These ICs induce kidney injury, and in the absence of disease-specific therapy, up to 40% of patients with IgAN progress to kidney failure. IgA1 with its clustered O-glycans is unique to humans, which hampered development of small-animal models of IgAN. Here, we used a model wherein engineered ICs (EICs) formed from human Gd-IgA1 and recombinant human IgG autoantibody are injected into nude mice to induce glomerular injury mimicking human IgAN. In this model, we assessed the protective effects of sparsentan, a single-molecule dual endothelin angiotensin receptor antagonist (DEARA) versus vehicle on EIC-induced glomerular proliferation and dysregulation of gene expression in the kidney. Oral administration of sparsentan (60 or 120 mg/kg daily) to mice intravenously injected with EIC attenuated the EIC-induced glomerular hypercellularity. Furthermore, analysis of changes in the whole kidney transcriptome revealed that key inflammatory and proliferative biological genes and pathways that are upregulated in this EIC model of IgAN were markedly reduced by sparsentan, including complement genes, integrin components, members of the mitogen-activated protein kinase family, and Fc receptor elements. Partial overlap between mouse and human differentially expressed genes in IgAN further supported the translational aspect of the immune and inflammatory components from our transcriptional findings. In conclusion, our data indicate that in the mouse model of IgAN, sparsentan targets immune and inflammatory processes leading to protection from mesangial hypercellularity.NEW & NOTEWORTHY The mechanisms by which deposited IgA1 immune complexes cause kidney injury during early phases of IgA nephropathy are poorly understood. We used an animal model we recently developed that involves IgA1-IgG immune complex injections and determined pathways related to the induced mesangioproliferative changes. Treatment with sparsentan, a dual inhibitor of endothelin type A and angiotensin II type 1 receptors, ameliorated the induced mesangioproliferative changes and the associated alterations in the expression of inflammatory genes and networks.

LIF/JAK2/STAT1 Signaling Enhances Production of Galactose-Deficient IgA1 by IgA1-Producing Cell Lines Derived From Tonsils of Patients With IgA Nephropathy.

Introduction: Galactose-deficient IgA1 (Gd-IgA1) plays a key role in the pathogenesis of IgA nephropathy (IgAN). Tonsillectomy has been beneficial to some patients with IgAN, possibly due to the removal of tonsillar cytokine-activated cells producing Gd-IgA1. To test this hypothesis, we used immortalized IgA1-producing cell lines derived from tonsils of patients with IgAN or obstructive sleep apnea (OSA) and assessed the effect of leukemia inhibitory factor (LIF) or oncostatin M (OSM) on Gd-IgA1 production. Methods: Gd-IgA1 production was measured by lectin enzyme-linked immunosorbent assay; JAK-STAT signaling in cultured cells was assessed by immunoblotting of cell lysates; and validated by using small interfering RNA (siRNA) knock-down and small-molecule inhibitors. Results: IgAN-derived cells produced more Gd-IgA1 than the cells from patients with OSA, and exhibited elevated Gd-IgA1 production in response to LIF, but not OSM. This effect was associated with dysregulated STAT1 phosphorylation, as confirmed by STAT1 siRNA knock-down. JAK2 inhibitor, AZD1480 exhibited a dose-dependent inhibition of the LIF-induced Gd-IgA1 overproduction. Unexpectedly, high concentrations of AZD1480, but only in the presence of LIF, reduced Gd-IgA1 production in the cells derived from patients with IgAN to that of the control cells from patients with OSA. Based on modeling LIF-LIFR-gp130-JAK2 receptor complex, we postulate that LIF binding to LIFR may sequester gp130 and/or JAK2 from other pathways; and when combined with JAK2 inhibition, enables full blockade of the aberrant O-glycosylation pathways in IgAN. Conclusion: In summary, IgAN cells exhibit LIF-mediated overproduction of Gd-IgA1 due to abnormal signaling. JAK2 inhibitors can counter these LIF-induced effects and block Gd-IgA1 synthesis in IgAN.

Effects of paroxetine hydrochloride combined with idebenone on inflammatory factors and antioxidant molecules in treatment of depression after ischemic stroke.

Objectives: To evaluate the effects of paroxetine hydrochloride combined with idebenone on inflammatory factors and antioxidant molecules in the treatment of depression after ischemic stroke. Methods: Randomized controlled trial was adopted on 80 patients with depression after ischemic stroke were randomly divided into two groups, with 40 patients in each group at Xingtai Sanli Health Quannan Clinic from March 17, 2019 to December 20, 2021. Both groups were given basic treatment. On this basis, the control group was treated with paroxetine hydrochloride, while the study group was treated with paroxetine hydrochloride combined with idebenone. The clinical efficacy was evaluated using the Hamilton Rating Scale for Depression (HRSD) before and after treatment. Additionally, the difference in HRSD score after treatment and the improvement in inflammatory factors and antioxidant molecules were compared and analyzed between the two groups. Results: After treatment, the HRSD score of the study group was significantly improved compared with that of the control group (p= 0.00). The effective rate was 82.5% in the study group, which was significantly higher than 62.5% in the control group (p= 0.04). After treatment, TNF-a, CRP and IL-6 in the study group were significantly lower than those in the control group (p= 0.00). Serum SOD, TAC and CAT levels in the study group were significantly higher than those in the control group after treatment (SOD and TAC, p= 0.00; CAT, p= 0.01). The incidence of adverse reactions was 37.5% in the study group and 25% in the control group. Although the incidence of adverse reactions in the study group was higher than that in the control group, the difference was not statistically significant (p= 0.23). Conclusion: Paroxetine hydrochloride combined with idebenone in the treatment of depression after ischemic stroke can significantly improve HRSD score, enhance clinical efficacy, reduce the levels of inflammatory factors, and increase the levels of antioxidant factors, without a significant increase in adverse reactions. Therefore, it is a safe and effective treatment method.

Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor-Mediated AXL Phosphorylation.

RATIONALE & OBJECTIVE: Immunoglobulin A nephropathy (IgAN) is a common glomerular disease, with mesangial cell proliferation as a major feature. There is no disease-specific treatment. Platelet-derived growth factor (PDGF) contributes to the pathogenesis of IgAN. To better understand its pathogenic mechanisms, we assessed PDGF-mediated AXL phosphorylation in human mesangial cells and kidney tissue biopsy specimens. STUDY DESIGN: Immunostaining using human kidney biopsy specimens and in vitro studies using primary human mesangial cells. SETTING & PARTICIPANTS: Phosphorylation of AXL was assessed in cultured mesangial cells and 10 kidney-biopsy specimens from 5 patients with IgAN, 3 with minimal change disease, 1 with membranous nephropathy, and 1 with mesangioproliferative glomerulonephritis (GN). PREDICTOR: Glomerular staining for phospho-AXL in kidney biopsy specimens of patients with mesangioproliferative diseases. OUTCOMES: Phosphorylated AXL detected in biopsy tissues of patients with IgAN and mesangioproliferative GN and in cultured mesangial cells stimulated with PDGF. ANALYTIC APPROACH: t test, Mann-Whitney test, and analysis of variance were used to assess the significance of mesangial cell proliferative changes. RESULTS: Immunohistochemical staining revealed enhanced phosphorylation of glomerular AXL in IgAN and mesangioproliferative GN, but not in minimal change disease and membranous nephropathy. Confocal-microscopy immunofluorescence analysis indicated that mesangial cells rather than endothelial cells or podocytes expressed phospho-AXL. Kinomic profiling of primary mesangial cells treated with PDGF revealed activation of several protein-tyrosine kinases, including AXL. Immunoprecipitation experiments indicated association of AXL and PDGF receptor proteins. An AXL-specific inhibitor (bemcentinib) partially blocked PDGF-induced cellular proliferation and reduced phosphorylation of AXL and PDGF receptor and the downstream signals (AKT1 and ERK1/2). LIMITATIONS: Small number of kidney biopsy specimens to correlate the activation of AXL with disease severity. CONCLUSIONS: PDGF-mediated signaling in mesangial cells involves transactivation of AXL. Finding appropriate inhibitors to block PDGF-mediated transactivation of AXL may provide new therapeutic options for mesangioproliferative kidney diseases such as IgAN.

[Discussion on traditional Chinese medicine properties of Myrtus communis leaves based on literature analysis and Chinese medicine theory].

Myrtus communis is a traditional medicinal aromatic plant in the Mediterranean. At present, the plant has been introduced and cultivated in the southern part of China, and it is mostly used for ornamental or cosmetic purposes. Based on literature analysis and the theory of Chinese medicine, we discussed the medicinal parts and properties of M. communis in this paper to provide a theoretical basis for exploring the medicinal value of M. communis and its compatibility with traditional Chinese medicines. Literatures were searched from Web of Science(core collection), PubMed, CNKI, VIP and Wanfang by using the set conditions as key words. Then the obtained literatures were screened and classified. Finally, a total of 376 articles were included, consisting of 44 reviews, 54 germplasm resources, 78 chemical researches, 48 studies on application, extraction, or quality, 18 human trials, 132 pharmacological studies, and 2 safety studies. Based on literature analysis and theories of Chinese medicine, the leaves of M. communis were finally selected as the medicinal part of Chinese medicine, and the traditional Chinese medicine properties of M. communis leaves were deduced as pungent, bitter, and cool. The channel tropisms of M. communis leaves included lung, liver, and large intestine, with functions of detoxifying, resolving a mass, and insecticide. It was used for mouth sores, vaginal itching, hemorrhoids and warts, etc.; appropriate amount shall be applied for external use, and the decoction form shall be used for washing the affected parts; 3-12 g equivalent product shall be used in decoction, and this herb shall be put into the decoction in a later stage. The clarification of the medicinal parts of M. communis, and the determination of the Chinese medicine properties of M. communis leaves would lay a theoretical foundation for its compatibility and application with Chinese medicines, and can do more contribution to the medical and healthcare industry in our country.

[Research progress and discussion on traditional Chinese medicine properties of Kaempferia parviflora].

Kaempferiae Parviflorae Rhizoma is the dried rhizome of Kaempferia parviflora in Zingiberaceae. It is originated and widely distributed in Thailand and other tropical and subtropical regions, where it has been used as food and medicine for thousands of years. K. parviflora is also planted in Yunnan and other places of China, but its traditional Chinese medicine properties are not clear, which greatly limits its compatibility with traditional Chinese medicines. In this article, the English and Chinese literatures of K. parviflora were searched from Web of Science, PubMed, Scopus, CNKI, Wanfang, and VIP databases for research and analysis. The medicinal properties of K. parviflora were preliminarily discussed based on the theory of traditional Chinese medicine under the guidance of clinical application and research literatures. The traditional Chinese medicine properties of K. parviflora were inferred as follows: flat, acrid, sweet. The channel tropisms of K. parviflora included kidney, spleen, stomach, and liver. The function of K. parviflora included tonifying kidney to strengthen essence, tonifying Qi and invigorating spleen, soothing liver and relieving depression. K. parviflora was clinically applied for the diseases such as syndrome of kidney essence deficiency, sex apathy, deficiency of spleen Qi, lassitude and asthenia, a weary spirit, obesity, diabetes, liver Qi stagnation, depression, and restless. The equivalent of dry power is 1.5 g·d~(-1) and the equivalent of decoction is 1.5-6 g·d~(-1). The determination of traditional Chinese medicine properties of K. parviflora has indeed laid a theoretical foundation for its application in the field of traditional Chinese medicine and enriched traditional Chinese medicine resources.

Pgc-1α Promotes Phosphorylation, Inflammation, and Apoptosis in H9c2 Cells During the Early Stage of Lipopolysaccharide Induction.

Cardiac dysfunction in severe sepsis is associated with increased mortality. However, the molecular mechanisms underlying septic heart dysfunction remain unclear. Expression of peroxisome proliferator-activated receptor-γ coactivator 1α (Pgc-1α), concentrations of inflammatory factors, and activation of the nuclear factor kappa-B (NF-κB) signaling pathway were examined in H9c2 cells after a 24-h lipopolysaccharide (LPS) stimulation period using qPCR, enzyme-linked immunosorbent assays (ELISAs), and western blots (WBs), respectively. Pgc-1α was overexpressed and suppressed in cells using a lentivirus vector and siRNA, respectively. The effects of Pgc-1α dysfunction on the release of inflammatory factors and apoptosis were analyzed. Pgc-1α expression was increased after LPS induction for 0.5 h and returned to the pre-induction level at 2 h. Levels of IL-1ß, IL-6, and TNF-α increase after LPS induction for 0.5 h and accumulated in the culture supernatants over time. The WBs revealed the highest Pgc-1α and phospho (p)-p65 protein levels after LPS induction for 0.5 h, followed by a decrease; moreover, the cleaved-caspase-3 level increased after LPS induction for 0.5 h and increased gradually thereafter. A functional analysis of Pgc-1α revealed that overexpression of this protein enhanced LPS-induced inflammatory factors and p-p65 levels and inhibited apoptosis during the early stage after LPS induction (0.5 and 4 h). In contrast, the inhibition of Pgc-1α expression inhibited the LPS expression-associated increases in inflammatory factors and p-p65 and promoted apoptosis. Pgc-1α promoted LPS-induced p65 phosphorylation and inflammatory factor release while inhibiting apoptosis.

Experimental evidence of pathogenic role of IgG autoantibodies in IgA nephropathy.

BACKGROUND: IgA nephropathy is thought to be an autoimmune disease wherein galactose-deficient IgA1 (Gd-IgA1) is recognized by IgG autoantibodies, resulting in formation and renal accumulation of nephritogenic immune complexes. Although this hypothesis is supported by recent findings that, in renal immunodeposits of IgA nephropathy patients, IgG is enriched for Gd-IgA1-specific autoantibodies, experimental proof is still lacking. METHODS: IgG isolated from sera of IgA nephropathy patients or produced as a recombinant IgG (rIgG) was mixed with human Gd-IgA1 to form immune complexes. IgG from healthy individuals served as a control. Nude and SCID mice were injected with human IgG and Gd-IgA1, in immune complexes or individually, and their presence in kidneys was ascertained by immunofluorescence. Pathologic changes in the glomeruli were evaluated by quantitative morphometry and exploratory transcriptomic profiling was performed by RNA-Seq. RESULTS: Immunodeficient mice injected with Gd-IgA1 mixed with IgG autoantibodies from patients with IgA nephropathy, but not Gd-IgA1 mixed with IgG from healthy individuals, displayed IgA, IgG, and mouse complement C3 glomerular deposits and mesangioproliferative glomerular injury with hematuria and proteinuria. Un-complexed Gd-IgA1 or IgG did not induce pathological changes. Moreover, Gd-IgA1-rIgG immune complexes injected into immunodeficient mice induced histopathological changes characteristic of human disease. Exploratory transcriptome profiling of mouse kidney tissues indicated that these immune complexes altered gene expression of multiple pathways, in concordance with the changes observed in kidney biopsies of patients with IgA nephropathy. CONCLUSIONS: This study provides the first in vivo evidence for a pathogenic role of IgG autoantibodies specific for Gd-IgA1 in the pathogenesis of IgA nephropathy.

Leukemia Inhibitory Factor Signaling Enhances Production of Galactose-Deficient IgA1 in IgA Nephropathy.

OBJECTIVES: IgA nephropathy (IgAN) is thought to involve an autoimmune process wherein galactose-deficient IgA1 (Gd-IgA1), recognized as autoantigen by autoantibodies, forms pathogenic immune complexes. Mounting evidence has implicated abnormal activation of some protein-tyrosine kinases (PTKs) in IgAN. Furthermore, genome-wide association studies (GWAS) of IgAN provided insight into disease pathobiology and genetics. A GWAS locus on chromosome 22q12 contains genes encoding leukemia inhibitory factor (LIF) and oncostatin M, interleukin (IL)-6-related cytokines implicated in mucosal immunity and inflammation. We have previously shown that IL-6 mediates overproduction of Gd-IgA1 through aberrant STAT3 activation. Here, we show that LIF enhanced production of Gd-IgA1 in IgA1-secreting cells of patients with IgAN and provide initial analyses of LIF signaling. METHODS: We characterized LIF signaling that is involved in the overproduction of Gd-IgA1, using IgA1-secreting cell lines derived from peripheral blood of patients with IgAN and healthy controls (HC). We used global PTK activity profiling, immunoblotting, lectin ELISA, and siRNA knock-down. RESULTS: LIF stimulation did not significantly affect production of total IgA1 in IgA1-secreting cells from patients with IgAN or HC. However, LIF increased production of Gd-IgA1, but only in the cells from patients with IgAN. LIF stimulation enhanced phosphorylation of STAT1 in IgA1-secreting cells from patients with IgAN to a higher degree than in the cells from HC. siRNA knock-down of STAT1 blocked LIF-mediated overproduction of Gd-IgA1. Unexpectedly, this abnormal phosphorylation of STAT1 in IgA1-secreting cells from patients with IgAN was not mediated by JAK, but rather involved activation of Src-family PTKs (SFKs). CONCLUSION: Abnormal LIF/STAT1 signaling represents another pathway potentially leading to overproduction of Gd-IgA1 in IgAN, providing possible explanation for the phenotype associated with chromosome 22q12 GWAS locus. Abnormal LIF/STAT1 signaling and the associated SFKs may represent potential diagnostic and/or therapeutic targets in IgAN.

Assessment of inflammatory markers and mitochondrial factors in a rat model of sepsis-induced myocardial dysfunction.

The present study aimed to investigate the expression of inflammatory markers and mitochondrial function-related genes, as well as their temporal relationship with cardiac myocyte injury in a rat model of sepsis. The sepsis model was constructed using cecal ligation and puncture (CLP). Two hours after CLP, the levels of inflammatory cytokines (interleukin [IL]-1ß, IL-6, and TNFα) and myocardial function markers (serum brain natriuretic peptide [BNP], cardiac troponin-I [cTNI], and procalcitonin [PCT]) were increased significantly, falling from around 9 hours postoperatively. The concentration of nitric oxide (NO) in the heart tissue was increased 6 hours after CLP. The heart rate (HR) of rats that underwent CLP decreased 2 hours after surgery and then increased to above-normal values. The left ventricular short axis shortening (FS) and left ventricular ejection fraction (LVEF) were decreased at 2 hours postoperatively and reached a minima at 6 hours. Stroke volume (SV), cardiac output (CO), and changes and heart index (CI) results indicated myocardial dysfunction. Western blot analysis demonstrated the increased expression of mitochondrial function-related proteins and activation of mitochondrial apoptotic pathways. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays revealed that the proportion of proapoptotic cells was significantly higher in rats that underwent CLP than sham surgery at 2 to 24 hours postoperatively. Taken together, our results indicate that-in the rat model-CLP-induced sepsis leads to impaired cardiac function. Furthermore, induction of the expression of mitochondrial function-related genes indicated that myocardial cell mitochondrial function was disrupted, further aggravating cardiomyocyte apoptosis. These results provide a theoretical basis for the treatment of sepsis-induced myocardial dysfunction.

Glomerular Immunodeposits of Patients with IgA Nephropathy Are Enriched for IgG Autoantibodies Specific for Galactose-Deficient IgA1.

BACKGROUND: IgA nephropathy (IgAN) is the leading primary GN worldwide. The disease is thought to result from glomerular deposition of circulating immune complexes of IgG bound to galactose-deficient IgA1 (Gd-IgA1). However, routine immunofluorescence microscopy fails to detect IgG in many kidney biopsies from patients with IgAN and the specificity of IgG in immunodeposits has not been tested. METHODS: We used remnant frozen kidney-biopsy specimens from 34 patients with IgAN; 14 were IgG-positive and 20 were IgG-negative by routine immunofluorescence microscopy. Six patients with primary membranous nephropathy (MN) and eight with lupus nephritis (LN) served as controls. IgG in the kidney tissue was extracted and its amount determined by ELISA. IgG molecular integrity was assessed by SDS-PAGE immunoblotting. Antigenic specificity of extracted IgG was determined by ELISA using phospholipase A2 receptor (PLA2R) or Gd-IgA1 as antigen. In addition, ten other IgAN cases, six IgG-positive and four IgG-negative by routine immunofluorescence, were used for colocalization studies by confocal microscopy. RESULTS: IgG extracted from MN but not IgAN immunodeposits reacted with PLA2R. Conversely, IgG extracted from IgAN but not MN or LN immunodeposits reacted with Gd-IgA1. Even IgAN kidney-biopsy specimens without IgG by routine immunofluorescence microscopy had IgG specific for Gd-IgA1. Confocal microscopy confirmed the presence of IgG in the IgAN biopsies with colocalization of glomerular IgA and IgG. CONCLUSIONS: These results reveal for the first time that IgAN kidney biopsies, with or without IgG by routine immunofluorescence, contain Gd-IgA1-specific IgG autoantibodies. These findings support the importance of these autoantibodies in the pathogenesis of IgAN.

Defining HIV-1 Envelope N-Glycan Microdomains through Site-Specific Heterogeneity Profiles.

The HIV-1 envelope (Env) glycans shield the surface of Env from the immune system and form integral interactions important for a functional Env. To understand how individual N-glycosylation sites (NGS) coordinate to form a dynamic shield and evade the immune system through mutations, we tracked 20 NGS in Env from HIV-transmitted/founder (T/F) and immune escape variants and their mutants involving the N262 glycan. NGS were profiled in a site-specific manner using a high-resolution mass spectrometry (MS)-based workflow. Using this site-specific quantitative heterogeneity profiling, we empirically characterized the interdependent NGS of a microdomain in the high-mannose patch (HMP). The changes (shifts) in NGS heterogeneity between the T/F and immune escape variants defined a range of NGS that we further probed for exclusive combinations of sequons in the HMP microdomain using the Los Alamos National Laboratory HIV sequence database. The resultant sequon combinations, including the highly conserved NGS N262, N448, and N301, created an immune escape map of the conserved and variable sequons in the HMP microdomain. This report provides details on how some clustered NGS form microdomains that can be identified and tracked across Env variants. These microdomains have a limited number of N-glycan-sequon combinations that may allow the anticipation of immune escape variants.IMPORTANCE The Env protein of HIV is highly glycosylated, and the sites of glycosylation can change as the virus mutates during immune evasion. Due to these changes, the glycan location and heterogeneity of surrounding N-glycosylation sites can be altered, resulting in exposure of different glycan or proteoglycan surfaces while still producing a viable HIV variant. These changes present a need for vaccine developers to identify Env variants with epitopes most likely to induce durable protective responses. Here we describe a means of anticipating HIV-1 immune evasion by dividing Env into N-glycan microdomains that have a limited number of N-glycan sequon combinations.

Elevated Transient Receptor Potential Melastatin 8 (TRPM8) Expression Is Correlated with Poor Prognosis in Pancreatic Cancer.

BACKGROUND The transient receptor potential melastatin 8 (TRPM8) was found to be expressed abnormally in a variety of tumors and is associated with unfavorable prognosis in human cancers. However, its clinical significance in pancreatic cancer (PC) is mostly unknown. MATERIAL AND METHODS qRT-PCR was performed to measure the expression of TRPM8 in 110 pairs of PC tissues and the adjacent non-cancerous tissues. The association of TRPM8 expression with the clinical characters of PC patients was analyzed using the chi-square test. Furthermore, the prognostic value of TRPM8 was determined with Kaplan-Meier survival curve and Cox regression analysis. RESULTS We found that the expression level of TRPM8 was significantly elevated in PC tissues compared to the non-cancerous controls (P<0.001). In addition, a close relationship was observed between elevated TRPM8 expression with large tumor size (P=0.001), advanced TNM (P=0.013), and distant metastasis (P=0.034). Survival analysis suggested that patients with high TRPM8 expression has worse OS (P=0.001) and DFS (P<0.001) than those with low TRPM8 expression. Moreover, TRPM8 was confirmed as a valuable prognostic biomarker for OS (HR=1.913; 95% CI: 1.020-3.589; P=0.043) or DFS (HR=2.374; 95% CI: 1.269-4.443; P=0.007) of PC patients. CONCLUSIONS This study shows that TRPM8 expression is significantly up-regulated in PC and it might be a useful prognostic factor for patients with PC.

Antioxidant Activity and Hepatoprotective Potential of Quercetin 7-Rhamnoside In Vitro and In Vivo.

Hypericum japonicum is traditionally used as a folk medicine to treat cholestasis and hepatitis. Quercetin 7-rhamnoside (Q7R) is one of the main flavonoid components of Hypericum japonicum and has been rarely studied. The aim of the present study was to evaluate the antioxidant activity and hepatoprotective potential of Q7R. In the in vitro experiments, DPPH, ABTS and ferric reducing antioxidant power (FRAP) assays were first performed to assess the antioxidant properties of Q7R, and then a H2O2-induced oxidative damage cellular model was used to determine the cytoprotective and antioxidant properties of Q7R in human liver L-02 cells. In the in vivo experiment, the hepatoprotective activity of Q7R was evaluated by carbon tetrachloride (CCl4)-induced liver damage model in mice. The results of the three in vitro assays (DPPH, ABTS and FRAP) demonstrated that Q7R significantly exhibited antioxidant activity. The cell experiment results showed that Q7R possessed cytoprotective and antioxidant effects on H2O2-treated L-02 cells. In the in vivo experiments, Q7R suppressed the up-regulation of serum activities of ALT, AST, LDH and triglyceride (TG) levels with dose-dependency. Q7R down-regulated the production of MDA and increased the hepatic GSH content and antioxidant enzymes CAT activities. Hepatic morphological analysis was also performed to confirm the biochemical changes. In summary, these results suggested that Q7R could be considered as a potential source of natural antioxidants, and may become a promising candidate for the treatment of liver injury in the future.

Inhibition of STAT3 Signaling Reduces IgA1 Autoantigen Production in IgA Nephropathy.

INTRODUCTION: IgA nephropathy is a chronic renal disease characterized by mesangial immunodeposits that contain autoantigen, which is aberrantly glycosylated IgA1 with some hinge-region O-glycans deficient in galactose. Macroscopic hematuria during an upper respiratory tract infection is common among patients with IgA nephropathy, which suggests a connection between inflammation and disease activity. Interleukin-6 (IL-6) is an inflammatory cytokine involved in IgA immune response. We previously showed that IL-6 selectively increases production of galactose-deficient IgA1 in IgA1-secreting cells from patients with IgA nephropathy. METHODS: We characterized IL-6 signaling pathways involved in the overproduction of galactose-deficient IgA1. To understand molecular mechanisms, IL-6 signaling was analyzed by kinomic activity profiling and Western blotting, followed by confirmation assays using siRNA knock-down and small-molecule inhibitors. RESULTS: STAT3 was differentially activated by IL-6 in IgA1-secreting cells from patients with IgA nephropathy compared with those from healthy control subjects. Specifically, IL-6 induced enhanced and prolonged phosphorylation of STAT3 in the cells from patients with IgA nephropathy, which resulted in overproduction of galactose-deficient IgA1. This IL-6-mediated overproduction of galactose-deficient IgA1 could be blocked by small molecule inhibitors of JAK/STAT signaling. DISCUSSION: Our results revealed that IL-6-induced aberrant activation of STAT3-mediated overproduction of galactose-deficient IgA1. STAT3 signaling pathway may thus represent a new target for disease-specific therapy of IgA nephropathy.

A new method to evaluate the dose-effect relationship of a TCM formula Gegen Qinlian Decoction: "Focus" mode of integrated biomarkers.

It is difficult to accurately evaluate the efficacy of traditional Chinese medicine (TCM), which leads to the uncertainty and complexity of dose-effect analysis. In this study we established the "Focus" mode of biomarkers to characterize the dose-effect relationship of Gegen Qinlian Decoction (GQD), a TCM formula for treating type 2 diabetes mellitus (2-DM). A rat model of 2-DM was established through high fat diet feeding combined with low-dose STZ injection. Rats with 2-DM were administered high, middle or low doses (6.785, 4.071, 1.357 mg·kg-1·d-1, respectively) of GQD extract for 60 d. Metformin (300 mg·kg-1·d-1) was taken as the positive control. Blood samples were collected to assess serum biochemical indexes and metabolic profiling. After "Focus" analysis, the biochemical index triglycerides (TG) and insulin sensitivity (ISI) were identified as focused integrated biomarkers (FIBs), while arachidonic acid and docosatetraenoic acid were the metabolic FIBs. Dose-effect relationship curves of GQD were built based on these types of FIBs. Furthermore, the two dose-effect relationship curves showed similar trends with the middle dosage displaying the greatest efficacy, suggesting that insulin function and arachidonic acid metabolism played important roles in 2-DM and the responses to GQD. The metabolic FIB docosatetraenoic should be further explored for understanding its involvement in the process of 2-DM occurrence and the treatment. This "Focus" mode provides a novel strategy to evaluate the dose-effect relationship of a TCM. The system and concepts established here may also be applicable for assessing the dose-effect relationships of Western medicines.

Determination of gardenia yellow colorants in soft drink, pastry, instant noodles with ultrasound-assisted extraction by high performance liquid chromatography-electrospray ionization tandem mass spectrum.

A novel, rapid and simple analytical method was developed for the quantitative determination of crocin, crocetin and geniposide in soft drink, pastry and instant noodles. The solid samples were relatively homogenized into powders and fragments. The gardenia yellow colorants were successively extracted with methanol using ultrasound-assisted extraction. The analytes were quantitatively measured in the extracts by liquid chromatography coupled with electrospray ionization tandem mass spectrometry. High correlation coefficients (r(2)>0.995) of crocin, crocetin and geniposide were obtained within their linear ranges respectively (50-1000ng/mL, 50-1000ng/mL, 15-240ng/mL) by external standard method. The limits of detection (LODs) were 0.02µg/g for crocin, 0.01µg/g for crocetin and 0.002µg/g for geniposide. And the limits of quantitation (LOQs) were in the ranges of 0.05-0.45µg/g for crocin, and in the ranges of 0.042-0.32µg/g for crocetin, and in the ranges of 0.02-0.15µg/g for geniposide in soft drink, pastry and instant noodles samples. The average recoveries of crocin, crocetin and geniposide ranged from 81.3% to 117.6% in soft drink, pastry and instant noodles. The intra- and inter-day precisions were respectively in the range of 1.3-4.8% and 1.7-11.8% in soft drink, pastry and instant noodle. The developed methods were successfully validated and applied to the soft drink, pastry, and instant noodles collected from the located market in Beijing from China. Crocin, crocetin and geniposide were detected in the collected samples. The average concentrations ranged from 0.84 to 4.20mg/g for crocin, and from 0.62 to 3.11mg/g for crocetin, and from 0.18 to 0.79mg/g for gardenia in various food samples. The method can provide evidences for government to determine gardenia yellow pigments and geniposide in food.

Somatic Mutations Modulate Autoantibodies against Galactose-Deficient IgA1 in IgA Nephropathy.

Autoantibodies against galactose-deficient IgA1 drive formation of pathogenic immune complexes in IgA nephropathy. IgG autoantibodies against galactose-deficient IgA1 in patients with IgA nephropathy have a specific amino-acid sequence, Y1CS3, in the complementarity-determining region 3 of the heavy chain variable region compared with a Y1CA3 sequence in similar isotype-matched IgG from healthy controls. We previously found that the S3 residue is critical for binding galactose-deficient IgA1. To determine whether this difference is due to a rare germline sequence, we amplified and sequenced the corresponding germline variable region genes from peripheral blood mononuclear cells of seven patients with IgA nephropathy and six healthy controls from whom we had cloned single-cell lines secreting monoclonal IgG specific for galactose-deficient IgA1. Sanger DNA sequencing revealed that complementarity-determining region 3 in the variable region of the germline genes encoded the Y1C(A/V)3 amino-acid sequence. Thus, the A/V>S substitution in the complementarity-determining region 3 of anti-galactose-deficient-IgA1 autoantibodies of the patients with IgA nephropathy is not a rare germline gene variant. Modeling analyses indicated that the S3 hydroxyl group spans the complementarity-determining region 3 loop stem, stabilizing the adjacent ß-sheet and stem structure, important features for effective binding to galactose-deficient IgA1. Understanding processes leading to production of the autoantibodies may offer new approaches to treat IgA nephropathy.

New Insights into the Pathogenesis of IgA Nephropathy.

BACKGROUND: IgA nephropathy, a frequent cause of end-stage renal disease, is an autoimmune disease wherein immune complexes consisting of IgA1 with galactose-deficient O-glycans (autoantigen) and anti-glycan autoantibodies deposit in glomeruli and induce renal injury. Multiple genetic loci associated with disease risk have been identified. The prevalence of risk alleles varies geographically, highest in eastern Asia and northern Europe, fewer in other parts of Europe and North America, and the least in Africa. IgA nephropathy is diagnosed from pathological assessment of a renal biopsy specimen. Currently, therapy is not disease-targeted but rather is focused on maintaining control of blood pressure and proteinuria, ideally with suppression of angiotensin II. Possible additional approaches differ between countries. Disease-specific therapy as well as new tools for diagnosis, prognosis, and assessment of responses to therapy are needed. SUMMARY: Glycosylation pathways associated with aberrant O-glycosylation of IgA1 and, thus, production of autoantigen, have been identified. Furthermore, unique characteristics of the autoantibodies in IgA nephropathy have been uncovered. Many of these biochemical features are shared by patients with IgA nephropathy and Henoch-Schönlein purpura nephritis, suggesting that the two diseases may represent opposite ends of a spectrum of a disease process. Understanding the molecular mechanisms involved in formation of pathogenic IgA1-containing immune complexes will enable development of disease-specific therapies as well as diagnostic and prognostic biomarkers. KEY MESSAGES: IgA nephropathy is an autoimmune disease caused by glomerular deposition of nephritogenic circulating immune complexes consisting of galactose-deficient IgA1 (autoantigen) bound by anti-glycan autoantibodies. A better understanding of the multi-step process of pathogenesis of IgA nephropathy and the genetic and environmental contributing factors will lead to development of biomarkers to identify patients with progressive disease who would benefit from a future disease-specific therapy.

Outcomes of liver transplantation for end-stage biliary disease: A comparative study with end-stage liver disease.

AIM: To evaluate the outcomes of patients with end-stage biliary disease (ESBD) who underwent liver transplantation, to define the concept of ESBD, the criteria for patient selection and the optimal operation for decision-making. METHODS: Between June 2002 and June 2014, 43 patients with ESBD from two Chinese organ transplantation centres were evaluated for liver transplantation. The causes of liver disease were primary biliary cirrhosis (n = 8), cholelithiasis (n = 8), congenital biliary atresia (n = 2), graft-related cholangiopathy (n = 18), Caroli's disease (n = 2), iatrogenic bile duct injury (n = 2), primary sclerosing cholangitis (n = 1), intrahepatic bile duct paucity (n = 1) and Alagille's syndrome (n = 1). The patients with ESBD were compared with an end-stage liver disease (ESLD) case control group during the same period, and the potential prognostic values of multiple demographic and clinical variables were assessed. The examined variables included recipient age, sex, pre-transplant clinical status, pre-transplant laboratory values, operation condition and postoperative complications, as well as patient and allograft survival rates. Survival analysis was performed using Kaplan-Meier curves, and the rates were compared using log-rank tests. All variables identified by univariate analysis with P values < 0.100 were subjected to multivariate analysis. A Cox proportional hazard regression model was used to determine the effect of the study variables on outcomes in the study group. RESULTS: Patients in the ESBD group had lower model for end-stage liver disease (MELD)/paediatric end-stage liver disease (PELD) scores and a higher frequency of previous abdominal surgery compared to patients in the ESLD group (19.2 ± 6.6 vs 22.0 ± 6.5, P = 0.023 and 1.8 ± 1.3 vs 0.1 ± 0.2, P = 0.000). Moreover, the operation time and the time spent in intensive care were significantly higher in the ESBD group than in the ESLD group (527.4 ± 98.8 vs 443.0 ± 101.0, P = 0.000, and 12.74 ± 6.6 vs 10.0 ± 7.5, P = 0.000). The patient survival rate in the ESBD group was not significantly different from that of the ESBD group at 1, 3 and 5 years (ESBD: 90.7%, 88.4%, 79.4% vs ESLD: 84.9%, 80.92%, 79.0%, χ(2) = 0.194, P = 0.660). The graft-survival rates were also similar between the two groups at 1, 3 and 5 years (ESBD: 90.7%, 85.2%, 72.7% vs ESLD: 84.9%, 81.0%, 77.5%, χ(2) = 0.003, P = 0.958). Univariate analysis identified MELD/PELD score (HR = 1.213, 95%CI: 1.081-1.362, P = 0.001) and bleeding volume (HR = 0.103, 95%CI: 0.020-0.538, P = 0.007) as significant factors affecting the outcomes of patients in the ESBD group. However, multivariate analysis revealed that MELD/PELD score (HR = 1.132, 95%CI: 1.005-1.275, P = 0.041) was the only negative factor that was associated with short survival time. CONCLUSION: MELD/PELD criteria do not adequately measure the clinical characteristics and staging of ESBD. The allocation system based on MELD/PELD criteria should be re-evaluated for patients with ESBD.