Study on mechanism of salidroside against liver fibrosis by regulating CXCL16 / 中国中药杂志

In order to investigate the effect of salidroside on inhibiting liver fibrosis and its relationship with CXC chemokine ligand 16(CXCL16) in vivo and in vitro, totally 45 C57 BL/6 J male mice were randomly divided into normal group, model group and salidroside group, with 15 mice in each group. The mice in model group and salidroside group were injected intraperitoneally with 15% carbontetrachloride(CCl_4) olive oil solution to establish liver fibrosis model, and the mice in normal group were injected intraperitoneally with the same dose of olive oil. Salidroside group was given with 100 mg·kg~(-1 )salidroside by gavage, while the normal group and model group received the same amount of double distilled water by gavage. All mice were sacrificed after 5 weeks of intragastric administration. The pathological changes of mouse liver were observed by hematoxylin-eosin(HE) staining, and the degree of liver fibrosis was observed by sirius red staining. The protein expressions of collagen Ⅰ(ColⅠ), α-smooth muscle actin(α-SMA), fibronectin(FN), CXCL16, phosphorylated Akt(p-Akt), Akt in liver tissues were detected by Western blot. Hepatic stellate cell line JS 1 was cultured in vitro and divided into control group, model group(100 μg·L~(-1) CXCL16) and salidroside group(100 μg·L~(-1) CXCL16+1×10~(-5) mol·L~(-1) salidroside). Cell migration was detected by cell scratch, the mRNA expressions of ColⅠ and α-SMA were detected by RT-PCR, and the protein expressions of p-Akt and Akt were detected by Western blot. As compared with the normal group, the protein expressions of ColⅠ, α-SMA, FN, CXCL16, and p-Akt in the model group were significantly increased, and salidroside could reduce the expression of these indicators(P<0.05 or P<0.01). In vitro, CXCL16 could promote the migration of JS 1, increase the mRNA expressions of ColⅠ and α-SMA in JS 1, and enhance Akt phosphorylation in JS 1(P<0.05 or P<0.01). As compared with the model group, salidroside could inhibit the migration of JS 1 induced by CXCL16(P<0.05), and reduce the high expression of ColⅠ and α-SMA mRNA and the phosphorylation of Akt in JS 1 induced by CXCL16(P<0.05). In conclusion, salidroside might attenuate CCl_4-induced liver fibrosis in mice by inhibiting the migration, activation and Akt phosphorylation of hepatic stellate cells induced by CXCL16.

Chemokine axis CXCL16/CXCR6 in the progression of fibrosis / 中华微生物学和免疫学杂志

Fibrosis is the common pathological basis of various lesions. It is mainly manifested as excess collagen deposition and fibrous connective tissue in tissues with destructed structures and impaired function. At present, the pathogenesis of fibrosis is still unclear, and no effective treatment or prevention for fibrosis is available. In recent years, numerous studies have shown that chemokine CXCL16 and its receptor CXCR6 play an important role in the development of fibrosis. This paper summarizes the biological character-istics of CXCL16/CXCR6 axis and its role in fibrosis.

Autoantibodies against the second extracellular loop of the β1-adrenoceptor induced cardiac insuffi-ciency through up-regulating chemokine CXCL16 / 中华微生物学和免疫学杂志

Objective To screen out the cytokines relating to cardiac insufficiency caused by au-toantibodies against the second extracellular loop of the β1-adrenoceptor(β1-AA) using cytokine chip tech-nique, and to analyze the changes in signaling pathways. Methods Blood samples were collected from 67 patients with coronary artery disease(CAD) and 42 healthy subjects. ELISA was performed to detect β1-AA in plasma. BALB/c mice were passively immunized with the monoclonal antibodies against β1-AA (β1-AA mAb). Dynamic changes in mouse cardiac structure and functions were detected by heart ultrasound. Hema-toxylin and eosin (HE) and Masson staining were used to observe morphological changes in heart tissues.Cytokine chip technique was used to screen out the cytokines causing myocardial injury. Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis were used to classify the differentially expressed cytokines. Results Patients with CAD showed increased titer and posi-tive rate of β1-AA as compared with healthy subjects(P<0.001). The mouse model of heart injury was in-duced by β1-AA at the 8th week after immunization. A total of 37 differentially expressed cytokines were found in the model group,of which 11 cytokines were up-regulated and 26 cytokines were down-regulate as compared with those in the mouse control group. The level of CXCL16 was significantly increased in β1-AA-positive mice. GO analysis showed that CXCL16 was mainly involved in life processes including the positive regulation of cell death, migration, locomotion and cellular component movement. KEGG pathway enrich-ment analysis showed CXCL16 was significantly enriched in the pathway of cytokine-cytokine receptor inter-action and chemokine signaling pathway. ELISA showed that compared with β1-AA-negative patients, CXCL16 level was significantly increased in β1-AA-positive patients (P<0.01). A positive correlation was found between β1-AA and CXCL16 (P<0.01,r=0.43). Conclusion CXCL16 may play a critical role in the development of cardiac insufficiency induced by β1-AA.

Expression of CXCL16 /CXCR6 in fibroblast-like synoviocytes in rheumatoid arthritis and its role in synoviocyte proliferation / 北京大学学报(医学版)

Objective:It has been found that serum CXCL16 concentration in rheumatoid arthritis (RA) patients are significantly higher than those in osteoarthritis (OA) and normal subjects, and are positively correlated with disease activity and bone erosion.However, how is CXCL16 involved in the pathogenesis of RA is unclear.To evaluate the expression of CXCL16 and its receptor CXCR6 in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) patients, and to explore the role of CXCL16 in the proliferation of RA-FLS.Methods: FLS were isolated from knee synovial tissues obtained from 8 patients of RA, 7 osteoarthritis (OA) and 3 normal controls.The diagnosis of RA was in line with the 1987 American Rheumatology Association (ACR) RA classification criteria, osteoarthritis met the 1996 ACR revised knee osteoarthritis classification criteria.Control synovium were obtained from trauma caused knee joint injury in healthy individuals who required surgery.Human knee FLS were cultured by tissue explants adherent method.FLS between passages 3 and 5 were used in the experiment.Expression of CXCL16 and its receptor CXCR6 were performed in Western blot analysis.FLS proliferation follo-wing stimulation with TNF-α and different concentrations of CXCL16 was examined by cell counting kit-8 (CCK-8).Expression of phosphorylated AKT (pAKT) in RA-FLS stimulated by CXCL16 was quantified by Western blot.Different concentrations of recombinant human CXCL16 were added to the culture medium of RA-FLS.After 48 h culture, supernantants were collected, and TNF-α, IL-6, RANKL and MMP3 in culture supernatants of RA-FLS were determined by enzyme-linked immunosorbent assays (ELISA) operated following the kit instructions.Results: Expression of CXCL16 and CXCR6 in RA-FLS was significantly higher than that of OA and controls (P<0.05), but no significant difference was found between OA-FLS and control FLS.Proliferation of RA-FLS was markedly up-regulated after stimulation of CXCL16 (P <0.05).In the case of the CXCL16 stimulated OA-FLS and control FLS, the FLS proliferation remained basically unchanged.Expression of phosphorylated AKT in RA-FLS increased remarkably in condition of CXCL16 (50,100, 200 μg/L) stimulation.The levels of IL-6 and RANKL in culture supernatants of RA-FLS were obviously increased under CXCL16 (200 μg/L) stimulation, while TNF-α and MMP-3 levels in the culture supernatants remained unchanged after CXCL16 (200 μg/L) stimulation.Conclusion: This study shows that the expression of CXCL16 and its receptor was highly elevated in RA-FLS.Recombinant CXCL16 promoted RA-FLS proliferation and activation in vitro.All these indicate that CXCL16 play an important role in the pathogenesis of RA, anti-CXCL16 treatment may help to relieve inflammation and bone damage of RA patients.However, due to the limitations of this study, the role of CXCL16 and its receptors in RA-FLS remains to be elucidated by further research.

Effect of hypoxia on angiogenesis-related proteins in human dental pulp cells

Dental pulp is a highly vascularized tissue with high regenerative potential. Revascularization of severed vasculature in the tooth is required for pulp healing during avulsed tooth treatment. In this study, the relative expression of angiogenesis-related proteins was determined in human dental pulp cells using a human angiogenesis proteome profiler array. The proteome profiler array detected differentially expressed angiogenesis-related factors under conditions of hypoxia, which enhances the angiogenic potential of dental pulp cells. We confirmed that hypoxia regulates the mRNA expression of angiogenesis-related factors, including CXCL16 in dental pulp cells. Furthermore, conditioned media of hypoxic pulp cells induced tube-like structures of vascular endothelial cells, which were reduced by the neutralization of CXCL16 function. In conclusion, our data show that angiogenesis-related factors are differentially expressed by hypoxia in dental pulp cells and suggest that CXCL16 may involve in the revascularization of hypoxic dental pulp.

Activation of CXCL16 pathway by inflammation accelerates the progression of diabetic nephropathy / 中华肾脏病杂志

Objective To investigate the potential role of CXC chemokine ligand 16 (CXCL16)/CXC chemokine receptor 6 (CXCR6) pathway in the progression of diabetic nephropathy (DN). Methods 8?week old male db/db mice were randomly divided into DN group and DN inflamed group. 10% casein was subcutaneously injected to induce the DN mouse model with inflammation. In vitro, HK?2 cells were treated with high glucose (HG), and IL?1β+HG to investigate the effect of inflammatory stress on HK?2 cells. Further knockdown CXCL16 was mediated by RNA interference to determine the effects of CXCl16, then cells were divided into HG+IL?1βgroup, HG+IL?1β + siCXCL16 group and HG + IL?1β + vehicle group. Changes of renal function in mice were assessed by 24 h proteinuria and N?acetyl?β?D?glucosaminidase (NAG) during 8 weeks. The ultra?microstructure was checked by electron microscopy at 8th week. Lipid accumulation in kidneys and HK?2 were observed by Filipin staining and quantitative assay of intracellular free cholesterol. The protein expressions of CXCl16, CXCR6, a disintegrin and metalloproteinase?10 (ADAM10), fibronectin and α smooth muscle actin (α?SMA) in renal tissue were detected by immunohistochemistry and Western blotting. The mRNA and protein expressions of CXCl16, CXCR6, ADAM10, fibronectin andα?SMA in HK?2 cells were detected by real?time PCR and Western blotting, and protein expressions of CXCl16, CXCR6 and ADAM10 in HK?2 cells were also tested by cell immunofluorescence. Results Mice in DN inflamed group had higher 24 h proteinuria and NAG than those in DN group, and the differences between two groups shown statistical significance at 8th week (all P<0.05). Compared with DN mice, DN inflamed mice had more vacuoles within renal tubular cells, with mitochondrial swelling, deformation and decrease. Lipid accumulation and protein expressions of fibronectin and α?SMA were increased in DN inflamed group when compared with DN group (all P<0.05). Further, the expressions of CXCL16, CXCR6, ADAM10 were significantly increased in DN inflamed group (all P<0.05). In vitro, the mRNA and protein expressions of CXCL16, CXCR6, ADAM10, fibronectin and α?SMA, and lipid accumulation were increased in high glucose plus IL?1βgroup when compared with high glucose group (all P<0.05). However, after siRNA of CXCL16 transfection, the mRNA and protein expressions of CXCL16, CXCR6, ADAM10, fibronectin andα?SMA were down?regulated in HG+IL?1β+siCXCL16 group as compared with high glucose+IL?1βgroup (all P<0.05). Furthermore, lipid accumulation was decreased (P<0.05). Conclusion Inflammation accelerates tubulointerstitial injury in DN partly through the activation of CXCL16 pathway, which may facilitate the lipid accumulation in tubular epithelial cells.

CXCL16 deficiency attenuates STZ-induced diabetic nephropathy in mice / 中国病理生理杂志

AIM:To explore the effect of CXCL16 deficiency on streptozocin ( STZ)-induced diabetic nephrop-athy in mice.METHODS:CXCL16 knockout ( C16 KO) mice (8 years old) were used to build up diabetes model by treating with STZ.Age-and gender-matched wild-type ( WT) C57BL/6J mice treated with STZ were used as control.All mice were fed with chow diets for 12 weeks, and the development of diabetic nephropathy was evaluated.RESULTS:Compared with the WT mice treated with STZ, C16 KO mice treated with STZ presented lower fasting glucose levels and better glucose tolerance power.C16 KO mice treated with STZ also had lower urine protein levels and smaller areas of glo-merular injury as compared with WT mice treated with STZ.Furthermore, CXCL16 deficiency decreased the contents of re-nal reactive oxygen species ( ROS) , malondialdehyde ( MDA) and oxidized low-density lipoprotein ( ox-LDL) and the mR-NA expression of lectin-like oxidized low-density lipoprotein receptor 1 (Lox-1), and attenuated the expression of renal in-flammatory factors including tumor necrosis factor α( TNF-α) and interleukin 6 ( IL-6) , as well as chemokines including intercellular cell adhesion molecular 1 (ICAM-1) and chemokine C-X-C motif ligand 1 (CXCL1).CONCLUSION:CX-CL16 deficiency obviously inhibits the development of STZ-induced diabetic nephropathy in mice.

Joint action of CXCL16 and TNF-α genes polymorphisms in patients with atherosclerotic cerebral infarction / 实用医学杂志

Objective To investigate the joint action of CXCL16 G1850A and TNF-α T1031C genes polymorphisms in patients with atherosclerotic cerebral infarction (ACI). Methods CXCL16 gene, G1850A and TNF-α gene T1031C mononucleotide polymorphism were tested with polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) in 120 ACI patients and 75 healthy controls. Results The CXCL16 gene 1850 site AA genotype (35.8% vs 20.0%), A allele frequency (59.6% vs 44.0%), the TNF-αgene 1031 site CC genotype(2.5% vs 1.3%), C allele frequency(21.3% vs 11.3%)in ACI group were significantly higher than in the control group(P < 0.05). There was a positive correlation between the joint action of CXCL16 G1850A and TNF-α T1031C genes polymorphisms and ACI (χ2= 7.502,df = 2,P = 0.023). Conclusion The CXCL16 gene 1850, A allele and TNF-α gene 1031 C allele were risk factors for ACI. There is a positive correlation between the joint action of CXCL16 G1850A and TNF-α T1031C genes polymorphisms on ACI.

Role of the CXC12-CXCR4 Axis and CXCL16 in Inflammatory Bowel Disease

Numerous studies of colitis in IBD (inflammatory bowel diseases) patients and in animal models have demonstrated that both inflammatory cytokines and chemokines are up-regulated in settings of active inflammation. Blockade or absence of various cytokines and chemokines attenuates the disease in murine models of IBD. Therefore, identifying cytokines and chemokines involved in intestinal inflammation provide promising targets for the development of new drugs in the treatment of IBD. In general, chemokines have been implicated in many fundamental immune processes including lymphoid organogenesis, immune cell differentiation, development and positioning. Many chemokines are markedly increased in intestinal tissue from patients with IBD. In this study, we focused on the role of CXCL12-CXCR4 and CXCL16. CXCL12-CXCR4 axis plays a crucial role in the pathophysiology of IBD, especially UC, while SR-PSOX/CXCL16 plays a significant role in the pathophysiology of CD. Our present data suggest new insights into the etiology of IBD and we hope that the manipulation of these chemokines may have therapeutic value.

Prognostic Implications of the Expression of CXCL16 in Breast Carcinoma

BACKGROUND: Of the many prognostic factors for breast cancer, the relationship between an infiltration of inflammatory cells and the prognosis is debatable. Of the chemokines affecting cancer's inflammatory reactions, chemokine (C-X-C motif) ligand 16 (CXCL16) has attracted attention for its prognostic value in many cancers, including colorectal cancer and renal cell carcinoma. But the situation for breast carcinoma is unknown. The aim of this study was to examine the relationship between the prognostic factors and the CXCL16 expression in patients with breast carcinoma. METHODS: The patients (n=106) diagnosed with invasive ductal cancer of the breast were enrolled. We reviewed the clinicopathological factors of these patients, hematoxylin and eosin stains were prepared and estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2/neu) and CXCL16 immunostaining was performed. RESULTS: The ER expression was significantly correlated with age and inflammation. A CXCL16 expression was noted in 81.1% of the cases. No association was evident between a CXCL16 expression and any other parameter, including the survival rate. Multivariate analysis did not implicate ER, HER2/neu or CXCL16 as an independent prognostic factor, but the tumor size was independent predictive factor for the patient outcome. CONCLUSIONS: An inflammatory reaction mediated by CXCL16 is not associated with the prognosis of breast cancer or any clinicopathological factors.

Cxcl16 Interact With SARS-CoV N Protein In and Out Cell / 中国病毒学·英文版

Our study investigated the host cell protein which can interact with SARS-CoV N protein, and explored the functional connections. The eukaryotic expression vectors pEGFP-N1/SARS-CoVN and pdsRed2-N1/ CXCL16 were constructed and used to co-transfect HEK293FT cells by the calcium phosphate method. The HIS-tagged fusion protein SARS-CoVN-GFP was then built and purified for the binding assay in vitro. The co-localization of SARS-CoVN and CXCL16 in the cytoplasm of HEK293FT cells was also shown using confocal laser scanning microscopy. It is suggested that their interaction might be through direct combination. Under a fluorescence microscope, it was observed that the purified fusion protein SARS-CoVN-GFP was attached to the cell membrane of CXCL16-transfected cells, indicating that SARS-CoVN and CXCL16 can be mutually combined.

Relationship between serum CXCL16 levels and stroke subtypes in patients with acute cerebral infarction / 国际脑血管病杂志

Objective To investigate the changes of serum CXCL16 levels in patients with acute cerebral infarction and their relationship with the Trial of Org10172 in Acute Stroke Treatment (TOAST) etiological types of cerebral infarction. Methods The serum CXCL16 levels in 113 patients with acute cerebral infarction were measured by enzyme-linked immunosorbent assay (ELISA), and they were grouped according to TOAST types. The patients between all the subgroups and/or 32 healthy controls were compared. Results The serum CXCL16 levels in patient group were significantly higher than those in control group (2.29 ± 0.21 ng/mlvs.1.75±0.21 ng/ml, t= 12.863, P= 0.000); The serum CXCL16 levels in large artery atherosclerotic (LAA) stroke group were significantly higher than those in small artery occlusive (SAO) stroke group (2.38 ±0.23 ng/mL vs. 2.21 ±0.11 ng/ml, 1 =5. 743, P =0. 000), and both were significantly higher than those in the control group (q = 20. 501, P = 0. 000; q =13. 527, P= 0. 000). In the LAA group, there were no significant differences between the serum CXCL16 levels in ≥2 artery stenosis group and those in only 1 artery stenosis group (2.34 ±0.24 ng/ml vs. 2.46 ± 0. 19 ng/ml, t = - 1.969, P = 0. 054). Multivariate logistic regression analysis showed that CXCL16 (OR =0.972, 95% CI0.956-0. 978, P =0.001)and hyperlipidemia (OR =3.547, 95%CI 1.160-10. 848, P=0. 020) were the independent risk factors for cerebral infarction. Conclusions The serum CXCL16 levels increased in acute cerebral infarction, it closely related with the occurrence of cerebral infarction, and the LAA stroke group was significantly higher than the SAO stroke group.

The synthesis and shedding of transmembrane CXCL16 in first-trimester human trophoblast / 中华微生物学和免疫学杂志

Objective To detect the regulation of CXCL16 synthesis and shedding in first-trimester human trophoblasts. Methods Firstly, we analyzed the expression and secretion of chemokine CXCL16 in primary cultured trophoblasts by immunochemical staining and ELISA. Then we determined the soluble and cell-associated CXCLI6 respectively with and without treatments of cytokine IFN-γ, TNF-α, IL-4 and ADAM10 by ELISA. Results Trophoblast expressed and secreted CXCL16 in a stable level. Cytokine IFN-γ induced both synthesis and secretion of CXCL16 significantly ( P <0. 01 ) in trophoblasts. ADAM10 increased the shedding of chemokine domain of CXCL16 from trophoblasts but didn't influence the synthesis of CXCL16 protein in trophoblast. Conclusion IFN-γ and ADAM10 play important roles in production and shedding of transmembrane CXCL16 in first-trimester trophublasts.

Screen and Identification of The Protein-protein Interactors in The Host Cell With The SARS Coronavirus Nucleocapsid Protein / 生物化学与生物物理进展

15 SARS-CoV N Protein Interacting Protein (NPIP) were selected from host cells using Yeast Two-hybrid system (Y2H). These are Angiogenin, acyglycerol kinase, cytochrome oxydase subunit I, CXC chemokine ligand 16, epidermal growth factor receptor pathway substrate 15, glutathione S-transferase kappa 1,integrin beta 1, jun oncogene, NIMA (never in mitosis gene a)-related kinase 10, protein tyrosine kinase 2 beta,homo sapiens SH3KBP1 binding protein 1 and ubiquitin specific peptidase 53. With the aid of immunological co-precipitation (CO-IP), it was confirmed that chemokine CXCL16 was the interactor with SARS-CoV N protein in host cells.