G protein-coupled receptor kinase 2 as a novel therapeutic target for gland fibrosis of Sjögren's syndrome.

Sjogren's syndrome (SS) is a chronic, progressive autoimmune disorder characterized by gland fibrosis. We previously found a close correlation between gland fibrosis and the expression of G protein-coupled receptor kinase 2 (GRK2). In this study we explored the pathological and therapeutic significance of GRK2 in SS. Submandibular gland (SMG) antigen-induced SS mouse model was established in WT and GRK2+/- mice. We showed that the expression levels of GRK2 were significantly up-regulated in glandular tissue and positively correlated with fibrotic morphology in SS patients and mice. Hemizygous knockout of GRK2 significantly inhibited the gland fibrosis. In mouse salivary gland epithelial cells (SGECs), we demonstrated that GRK2 interacted with Smad2/3 to positively regulate the activation of TGF-ß-Smad signaling with a TGF-ß-GRK2 positive feedback loop contributing to gland fibrosis. Hemizygous knockout of GRK2 attenuated TGF-ß-induced collagen I production in SGECs in vitro and hindered gland fibrosis in murine SS though preventing Smad2/3 nuclear translocation. Around 28 days post immunization with SMG antigen, WT SS mice were treated with a specific GRK2 inhibitor paroxetine (Par, 5 mg·kg-1·d-1, i.g. for 19 days). We found that Par administration significantly attenuated gland fibrosis and alleviated the progression of SS in mice. We conclude that genetic knockdown or pharmacological inhibition of GRK2 significantly attenuates gland fibrosis and alleviates the progression of SS. GRK2 binds to Smad2/3 and positively regulates the activation of TGF-ß-Smad signaling. A TGF-ß-GRK2 positive feedback loop contributes to gland fibrosis. Our research points out that GRK2 could be a promising therapeutic target for treating SS.

Chronic ß-adrenergic stress contributes to cardiomyopathy in rodents with collagen-induced arthritis.

Patients with rheumatoid arthritis (RA) have a much higher incidence of cardiac dysfunction, which contributes to the high mortality rate of RA despite anti-arthritic drug therapy. In this study, we investigated dynamic changes in cardiac function in classic animal models of RA and examined the potential effectors of RA-induced heart failure (HF). Collagen-induced arthritis (CIA) models were established in rats and mice. The cardiac function of CIA animals was dynamically monitored using echocardiography and haemodynamics. We showed that cardiac diastolic and systolic dysfunction occurred in CIA animals and persisted after joint inflammation and that serum proinflammatory cytokine (IL-1ß, TNF-α) levels were decreased. We did not find evidence of atherosclerosis (AS) in arthritic animals even though cardiomyopathy was significant. We observed that an impaired cardiac ß1AR-excitation contraction coupling signal was accompanied by sustained increases in blood epinephrine levels in CIA rats. Furthermore, serum epinephrine concentrations were positively correlated with the heart failure biomarker NT-proBNP in RA patients (r2 = +0.53, P < 0.0001). In CIA mice, treatment with the nonselective ßAR blocker carvedilol (2.5 mg·kg-1·d-1, for 4 weeks) or the specific GRK2 inhibitor paroxetine (2.5 mg·kg-1·d-1, for 4 weeks) effectively rescued heart function. We conclude that chronic and persistent ß-adrenergic stress in CIA animals is a significant contributor to cardiomyopathy, which may be a potential target for protecting RA patients against HF.

Regulating Th17/Treg Balance Contributes to the Therapeutic Effect of Ziyuglycoside I on Collagen-Induced Arthritis.

To investigate the therapeutic effect and primary pharmacological mechanism of Ziyuglycoside I (Ziyu I) on collagen-induced arthritis (CIA) mice. CIA mice were treated with 5, 10, or 20 mg/kg of Ziyu I or 2 mg/kg of methotrexate (MTX), and clinical manifestations, as well as pathological changes, were observed. T cell viability and subset type were determined, and serum levels of transforming growth factor-beta (TGF-ß) and interleukin-17 (IL-17) were detected. The mRNA expression of retinoid-related orphan receptor-γt (RORγt) and transcription factor forkhead box protein 3 (Foxp3) in mouse spleen lymphocytes was ascertained by the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Molecular docking was used to detect whether there was a molecular interaction between Ziyu I and protein kinase B (Akt). The activation of mechanistic target of rapamycin (mTOR) in T cells was verified by Western blotting or immunofluorescence. Ziyu I treatment effectively alleviated arthritis symptoms of CIA mice, including body weight, global score, arthritis index, and a number of swollen joints. Similarly, pathological changes of joints and spleens in arthritic mice were improved. The thymic index, T cell activity, and RORγt production of Ziyu I-treated mice were significantly reduced. Notably, through molecular docking, western blotting, and immunofluorescence data analysis, it was found that Ziyu I could interact directly with Akt to reduce downstream mTOR activation and inhibit helper T cell 17 (Th17) differentiation, thereby regulating Th17/regulatory T cell (Treg) balance and improving arthritis symptoms. Ziyu I effectively improves arthritic symptoms in CIA mice by inhibiting mTOR activation, thereby affecting Th17 differentiation and regulating Th17/Treg balance.

87 Sr/86 Sr isotope ratios in rocks determined using inductively coupled plasma tandem mass spectrometry in O2 mode without prior Sr purification.

RATIONALE: An inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) instrument can be developed to determine 87 Sr/86 Sr ratios with an external precision better than 0.05% relative standard deviation (RSD) in "mass shift" mode without prior Sr purification. Previous studies suggested using CH3 F, N2 O, and SF6 as reaction gases for this method because a better reaction rate can be achieved with Sr+ than with O2 in the reaction cell. However, these gases are not commonly used in general chemistry laboratories, and processes using these gases are difficult to implement quickly due to regulations. We aim to develop a rapid method that can be applied to many samples for the accurate determination of 87 Sr/86 Sr isotope ratios with precision below 0.1% RSD (or approximately to the fourth decimal place). METHODS: We evaluated the accuracy and precision of 87 Sr/86 Sr ratios in certified reference materials and different rock types determined using ICP-MS/MS with O2 as the reaction gas in comparison with those determined using the multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS) method. RESULTS: This study showed that by using the ICP-MS/MS method, the 87 Sr/86 Sr ratios of BCR-2 and BHVO-2 do not vary significantly with and without prior Sr purification; when the Sr concentration of the measured solution is within the range of 60-350 ng/mL, there is no significant effect on the measured 87 Sr/86 Sr ratios. The results also showed that the 87 Sr/86 Sr ratios of 23 different rock types measured by ICP-MS/MS and MC-ICP-MS methods agree very well. CONCLUSIONS: The precision of the 87 Sr/86 Sr ratio measured using ICP-MS/MS varies between 0.0001 and 0.0019 (2SD). This precision is less than that of the MC-ICP-MS method but is sufficient for certain applications, such as identifying 87 Sr/86 Sr ratios in different rock types. These results suggest that the developed ICP-MS/MS method has the potential for future studies involving the identification of Sr sources.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis.

Hyperplasia and migration of fibroblast-like synoviocytes (FLSs) are the key drivers in the pathogenesis of rheumatoid arthritis (RA) and joint destruction. Abundant Yes-associated protein (YAP), which is a powerful transcription co-activator for proliferative genes, was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms. Using Gene Expression Omnibus database analysis, it was found that Salvador homolog-1 (SAV1), the pivotal negative regulator of the Hippo-YAP pathway, was slightly downregulated in RA synovium. However, SAV1 protein expression is extremely reduced. Subsequently, it was revealed that SAV1 is phosphorylated, ubiquitinated, and degraded by interacting with an important serine-threonine kinase, G protein-coupled receptor (GPCR) kinase 2 (GRK2), which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2 (PGE2) in RA. This process further contributes to the decreased phosphorylation, nuclear translocation, and transcriptional potency of YAP, and leads to aberrant FLSs proliferation. Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration. Similarly, paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations. Collectively, these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

Functions of G protein-coupled receptor 56 in health and disease.

Human G protein-coupled receptor 56 (GPR56) is encoded by gene ADGRG1 from chromosome 16q21 and is homologously encoded in mice, at chromosome 8. Both 687 and 693 splice forms are present in humans and mice. GPR56 has a 381 amino acid-long N-terminal extracellular segment and a GPCR proteolysis site upstream from the first transmembrane domain. GPR56 is mainly expressed in the heart, brain, thyroid, platelets, and peripheral blood mononuclear cells. Accumulating evidence indicates that GPR56 promotes the formation of myelin sheaths and the development of oligodendrocytes in the cerebral cortex of the central nervous system. Moreover, GPR56 contributes to the development and differentiation of hematopoietic stem cells, induces adipogenesis, and regulates the function of immune cells. The lack of GPR56 leads to nervous system dysfunction, platelet disorders, and infertility. Abnormal expression of GPR56 is related to the malignant transformation and tumor metastasis of several cancers including melanoma, neuroglioma, and gastrointestinal cancer. Metabolic disorders and cardiovascular diseases are also associated with dysregulation of GPR56 expression, and GPR56 is involved in the pharmacological resistance to some antidepressant and cancer drug treatments. In this review, the molecular structure, expression profile, and signal transduction of GPR56 are introduced, and physiological and pathological functions of GRP56 are comprehensively summarized. Attributing to its significant biological functions and its long N-terminal extracellular region that interacts with multiple ligands, GPR56 is becoming an attractive therapeutic target in treating neurological and hematopoietic diseases.

A Simplified Method Using a Single N,N,N',N'-Tetraoctyl Diglycolamide Resin Column for the Purification of Sr, Nd and Hf in Geological Materials and the Determination of Their Isotopic Ratios by Multi-collector Inductively Coupled Plasma-Mass Spectrometry.

A simplified method using a single column of N,N,N',N'-tetraoctyl diglycolamide (TODGA) resin is developed for the separation of Sr, Nd and Hf with matrix and interference elements from geological samples, and for subsequent determination of their isotopic ratios by multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS). The analytes of Sr, Nd and Hf are absorbed by the TODGA resin and eluted with 6 mol/L HNO3, 1.2 mol/L HCl and 1 mol/L HNO3-1.6 mol/L HF, respectively. The separation procedure is validated by the certified reference materials (CRMs) of BHVO-2, BCR-2 and AGV-2 with analyte recovery greater than 97%. The ratios are measured for 87Sr/86Sr, 143Nd/144Nd and 176Hf/177Hf and the mean values (2σ) are 0.703455 (16), 0.512977 (12) and 0.283108 (8) for BHVO-2, 0.705008 (18), 0.512633 (10) and 0.282878 (4) for BCR-2, and 0.703989 (20), 0.512791 (8) and 0.282982 (8) for AGV-2, which are consistent with the certified values.