Youguiwan Regulates CXCL8/CXCR1/2 Signaling Pathway and Ang-1 and Ang-2 Expression to Promote Ovarian Angiogenesis in Rats / 中国实验方剂学杂志

ObjectiveTo study the effects of Chinese herbal compound Youguiwan on angiogenesis of rats with ovarian dysfunction caused by natural aging and its relationship with chemokine interleukin 8 （CXCL8）/CXC chemokine receptor 1/2 （CXCR1/2） signaling pathway， angiopoietin 1 （Ang-1）， and angiopoietin 2 （Ang-2）， so as to explore its mechanism in improving the ovarian function. MethodFifty six female SD rats were randomly divided into the young control group （n=8） and modeling group （n=48， ovarian dysfunction caused by natural aging）. Rats in both the young control and modeling groups were routinely fed， during which the ones in the modeling group underwent exfoliative cytology of vaginal smears for five to seven days. The ones presented with prolonged estrous cycle， followed by continuous estrus and repeated pseudopregnancy revealed by vaginal cytology during four consecutive estrous cycles indicated early aging， and the young rats with keratinocyte proliferation index higher than 50% for 10 consecutive days were classified into the young control group. The successfully modeled rats were randomly divided into the early-aged group， estrogen （65 μg·kg-1·d-1） group， Zuoguiwan （33 g·kg-1·d-1） group， as well as the low-， medium-， and high-dose （1.2， 2.4， 4.8 g·kg-1·d-1） Youguiwan groups. Rats in the young control group and the early-aged group were gavaged with the same volume of normal saline for 30 days. After the experiment， the morphological changes in rat ovary were observed by hematoxylin-eosin （HE） staining. The protein expression levels of chemokines CXCL8， CXCR1， CXCR2， Ang-1， and Ang-2 in rat ovary were detected by Western blotting and immunohistochemistry， and the mRNA expression levels of CXCL8， CXCR1， CXCR2， Ang-1， and Ang-2 by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the young control group， the early-aged group exhibited reduced number of growing follicles， corpus luteum， and blood vessels at all levels， elevated atretic follicles （P<0.01）， up-regulated protein and mRNA expression of CXCL8， CXCR1， and CXCR2 in the ovarian tissue （P<0.01）， and down-regulated Ang-1 and Ang-2 protein and mRNA expression （P<0.05）. Compared with the early-aged group， each medication remarkably increased the number of growing follicles， corpus luteum， and blood vessels （P<0.05）， lowered the number of atretic follicles （P<0.05）， down-regulated the protein and mRNA expression levels of CXCL8， CXCR1， and CXCR2 in the ovarian tissue （P<0.05）， and up-regulated the protein and mRNA expression levels of Ang-1 and Ang-2 （P<0.05）. ConclusionYouguiwan down-regulates the levels of CXCL8， CXCR1， and CXCR2 in rat ovary and up-regulates the levels of Ang-1 and Ang-2 to promote ovarian angiogenesis and improve rat ovarian function.

In silico prediction of the functional and structural consequences of the non-synonymous single nucleotide polymorphism A122V in bovine CXC chemokine receptor type 1 / Predição in silico das consequências funcionais e estruturais do polimorfismo de nucleotídeo único não-sinônimo A122V no receptor de quimiocina CXC do tipo I bovino

Abstract The current study aimed to assess whether the A122V causal polymorphism promotes alterations in the functional and structural proprieties of the CXC chemokine receptor type 1 protein (CXCR1) of cattle Bos taurus by in silico analyses. Two amino acid sequences of bovine CXCR1 was selected from database UniProtKB/Swiss-Prot: a) non-polymorphic sequence (A7KWG0) with alanine (A) at position 122, and b) polymorphic sequence harboring the A122V polymorphism, substituting alanine by valine (V) at same position. CXCR1 sequences were submitted as input to different Bioinformatics' tools to examine the effects of this polymorphism on functional and structural stabilities, to predict eventual alterations in the 3-D structural modeling, and to estimate the quality and accuracy of the predictive models. The A122V polymorphism exerted tolerable and non-deleterious effects on the polymorphic CXCR1, and the predictive structural model for polymorphic CXCR1 revealed an alpha helix spatial structure typical of a receptor transmembrane polypeptide. Although higher variations in the distances between pairs of amino acid residues at target-positions are detected in the polymorphic CXCR1 protein, more than 97% of the amino acid residues in both models were located in favored and allowed conformational regions in Ramachandran plots. Evidences has supported that the A122V polymorphism in the CXCR1 protein is associated with increased clinical mastitis incidence in dairy cows. Thus, the findings described herein prove that the replacement of the alanine by valine amino acids provokes local conformational changes in the A122V-harboring CXCR1 protein, which could directly affect its post-translational folding mechanisms and biological functionality.

Resumo O presente estudo objetivou avaliar se o polimorfismo causal A122V promove alterações nas propriedades funcionais e estruturais da proteína receptora de quimiocina CXC do tipo 1 (CXCR1) de bovino Bos taurus por análises in silico. Duas sequências de aminoácidos da CXCR1 bovina foram selecionadas a partir do banco de dados UniProtKB/Swiss-Prot: a) sequência não-polimórfica (A7KWG0) contendo alanina (A) na posição 122, e b) sequência polimórfica carreando o polimorfismo A122V, causando a substituição de alanina por valina (V) na mesma posição. As sequências CXCR1 foram analisadas por diferentes ferramentas de Bioinformática para examinar o efeito desse polimorfismo sobre sua estabilidade, função e estrutura, predizer eventuais alterações na sua modelagem estrutural 3-D, bem como estimar a qualidade dos modelos preditos. O polimorfismo A122V exerceu efeitos toleráveis e não-deletérios sobre a CXCR1 polimórfica, apresentando um modelo estrutural de alfa-hélice típico de uma proteína receptora transmembranar para ambas as proteínas. Embora maiores variações nas distâncias entre os pares de aminoácidos nas posições-alvo tenham sido detectadas na proteína polimórfica, mais do que 97% dos aminoácidos em ambos os modelos foram situados em regiões ditas favoráveis e permitidas nos diagramas de Ramachandran. Evidências sustentam que o polimorfismo de nucleotídeo único A122V na proteína receptora CXCR1 está associado à aumentada incidência de mastite clínica em vacas leiteiras. Assim, as descobertas descritas aqui comprovam que a substituição do aminoácido alanina por valina provoca mudanças conformacionais locais na proteína CXCR1 polimórfica, que podem estar diretamente afetando seus mecanismos de enovelamento pós-traducionais e sua função biológica.

Clinical significance of the interleukin 8 and interleukin receptor 1 axis in stomach neoplasms / 中华老年医学杂志

Objective To explore sensitive indicators for the initiation,development,and metastasis of gastric cancer and to provide objective evidence for the early diagnosis,treatment,and progression monitoring of gastric cancer.Methods A total of 108 patients with gastric cancer were enrolled in this study.The expression of interleukin receptor 1 (CXCR1)in samples from gastric cancer and adjacent tissues was detected by immunohistochemistry and patient clinical data were collected for correlation analysis.Logistic regression analysis of the 5-year survival rate of patients was conducted.Results The positive CXCR1 expression rate in gastric neoplasm tissues was significantly higher than that in adjacent tissues.Nevertheless,CXCR1 was correlated with tumor differentiation (P =0.017),TNM staging (P =0.006),and the existence of lymphatic metastasis (P =0.035).The overall survival rate (P =0.043) and recurrence-free survival rate (P=0.029) of patients with positive CXCR1 were lower than those of patients with negative CXCR1.Conclusions CXCR1 expression levels increase in gastric neoplasm tissues and are associated with tumor differentiation,TNM staging,and lymphatic metastasis.Positive CXCR1 is correlated with poor prognosis and has the potential to serve as one of clinical prognostic indicators.

In silico prediction of the functional and structural consequences of the non-synonymous single nucleotide polymorphism A122V in bovine CXC chemokine receptor type 1

Abstract The current study aimed to assess whether the A122V causal polymorphism promotes alterations in the functional and structural proprieties of the CXC chemokine receptor type 1 protein (CXCR1) of cattle Bos taurus by in silico analyses. Two amino acid sequences of bovine CXCR1 was selected from database UniProtKB/Swiss-Prot: a) non-polymorphic sequence (A7KWG0) with alanine (A) at position 122, and b) polymorphic sequence harboring the A122V polymorphism, substituting alanine by valine (V) at same position. CXCR1 sequences were submitted as input to different Bioinformatics tools to examine the effects of this polymorphism on functional and structural stabilities, to predict eventual alterations in the 3-D structural modeling, and to estimate the quality and accuracy of the predictive models. The A122V polymorphism exerted tolerable and non-deleterious effects on the polymorphic CXCR1, and the predictive structural model for polymorphic CXCR1 revealed an alpha helix spatial structure typical of a receptor transmembrane polypeptide. Although higher variations in the distances between pairs of amino acid residues at target-positions are detected in the polymorphic CXCR1 protein, more than 97% of the amino acid residues in both models were located in favored and allowed conformational regions in Ramachandran plots. Evidences has supported that the A122V polymorphism in the CXCR1 protein is associated with increased clinical mastitis incidence in dairy cows. Thus, the findings described herein prove that the replacement of the alanine by valine amino acids provokes local conformational changes in the A122V-harboring CXCR1 protein, which could directly affect its post-translational folding mechanisms and biological functionality.

Resumo O presente estudo objetivou avaliar se o polimorfismo causal A122V promove alterações nas propriedades funcionais e estruturais da proteína receptora de quimiocina CXC do tipo 1 (CXCR1) de bovino Bos taurus por análises in silico. Duas sequências de aminoácidos da CXCR1 bovina foram selecionadas a partir do banco de dados UniProtKB/Swiss-Prot: a) sequência não-polimórfica (A7KWG0) contendo alanina (A) na posição 122, e b) sequência polimórfica carreando o polimorfismo A122V, causando a substituição de alanina por valina (V) na mesma posição. As sequências CXCR1 foram analisadas por diferentes ferramentas de Bioinformática para examinar o efeito desse polimorfismo sobre sua estabilidade, função e estrutura, predizer eventuais alterações na sua modelagem estrutural 3-D, bem como estimar a qualidade dos modelos preditos. O polimorfismo A122V exerceu efeitos toleráveis e não-deletérios sobre a CXCR1 polimórfica, apresentando um modelo estrutural de alfa-hélice típico de uma proteína receptora transmembranar para ambas as proteínas. Embora maiores variações nas distâncias entre os pares de aminoácidos nas posições-alvo tenham sido detectadas na proteína polimórfica, mais do que 97% dos aminoácidos em ambos os modelos foram situados em regiões ditas favoráveis e permitidas nos diagramas de Ramachandran. Evidências sustentam que o polimorfismo de nucleotídeo único A122V na proteína receptora CXCR1 está associado à aumentada incidência de mastite clínica em vacas leiteiras. Assim, as descobertas descritas aqui comprovam que a substituição do aminoácido alanina por valina provoca mudanças conformacionais locais na proteína CXCR1 polimórfica, que podem estar diretamente afetando seus mecanismos de enovelamento pós-traducionais e sua função biológica.

Expression and Clinical Significance of CXCR1 and CXCR2 in Triple-negative Breast Cancer / 中国医科大学学报

Objective To investigate the expression of CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2) in triplenegative breast cancer (TNBC) and its significance.Methods The EnVision immunohistochemistry system was used to detect the expression of CXCR1 and CXCR2 in 28 specimens from normal tissues adjacent to breast cancer,30 specimens of invasive ductal carcinoma not otherwise specified (IDC-NOS),and 34 TNBC specimens.The relationship between the expression of CXCR1 and CXCR2,and clinical pathological features was analyzed.Results In the adjacent normal tissues of breast cancer,the positive expression rates of CXCR 1 and CXCR2 were 89.3％ and 92.9％,respectively;CXCR1 and CXCR2 expression displayed no significant association with clinicopathological parameters such as age,tumor size,or Scarff-Bloom-Richardson (SBR) score.The positive expression rates of CXCR1 and CXCR2 were 63.3％ and 60.0％,respectively,in the IDC-NOS samples and 23.5％ and 35.3％,respectively,in the TNBC samples.There was a positive correlation between CXCR1 and CXCR2 expression in the TNBC samples (r =0.606,P ＜ 0.001).Conclusion Correlation of CXCR1 and CXCR2 expression with sample type was strong in adjacent normal tissues,moderate in IDC-NOS samples,and low in TNBC samples.CXCR1 expression was positively correlated with CXCR2 expression in all sample types.The expression of CXCR1 or CXCR2 was closely related to the development and promotion of TNBC.

Expressions and clinical significance of CXC chemokine receptor 1 and 2 and CXCL8 in hepatitis B related hepatocellular carcinoma / 中国免疫学杂志

Objective:To investigate the expression levels of CXCR1,CXCR2 and their common ligand CXCL8 in peripheral blood mononuclear cells (PBMCs) and liver biopsy from the patients with hepatitis B related hepatocellular carcinoma and their clinical significances.Methods:Quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the mRNA levels of CXCR1,CXCR2,CXCL8 in the peripheral blood mononuclear cells of thirty-six hepatitis B related hepatocellular carcinoma and the protein levels of CXCR1 and CXCR2 and CXCL8 in liver biopsy were detected by SP immunohistochemical method.The level of C-reactive protein in serum was determined by chemiluminescence immunoassay respectively.Then,the correlations between CRP and the mRNA of CXCR1,CXCR2 and CXCL8 were analyzed.Results:The mRNA levels of CXCR1 (0.952 7±0.197 2),CXCR2 (0.896 9±0.173 0),CXCL8 (1.771 9±0.248 9) in the PBMCs of hepatitis B related hepatocellular carcinoma were significantly higher than those in controls (P＜0.01).And the protein levels of CXCR1,CXCR2 and CXCL8 were also obviously increased in liver biopsy of hepatitis B related hepatocellular carcinoma (P＜0.05).In addition,there was positive correlations between the level of serum C-reactive protein and the mRNA expression of CXCR1 (r =0.54,P＜0.01),CXCR2 (r =0.49,P＜0.01),CXCL8 (r =0.63,P＜0.01).Conclusion:The levels of CXCR1,CXCR2 and CXCL8 significantly increased in hepatitis B related hepatocellular carcinoma patients and positively correlated with serum CRP,suggesting that CXCR1,CXCR2 and their common ligand CXCL8 signal transduction process may play a key role in the pathological process of hepatitis B related hepatocellular carcinoma,which may provide a new direction for the immune intervention therapy of hepatocellular carcinoma.

Expression and clinical significance of CXC chemokine receptors 1 and 2 and CXCL8 in primary hepatocellular carcinoma / 中华微生物学和免疫学杂志

Objective To investigate the expression and clinical significance of CXC chemokine receptors 1 and 2 (CXCR1 and CXCR2) and CXCL8 in peripheral blood mononuclear cells (PBMCs) and liver biopsy tissues from patients with primary hepatocellular carcinoma (PHC).Methods Serum specimens were collected from 36 patients with PHC, 30 patients with liver cirrhosis and 28 healthy subjects.Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the expression of CXCR1, CXCR2 and CXCL8 at mRNA level in PBMCs.Streptavidin-perosidase (SP) immunohistochemistry was used to detect the expression of CXCR1, CXCR2 and CXCL8 at protein level in liver biopsy tissues.Levels of C-reactive protein (CRP), alpha-fetoprotein (AFP) and ferritin (FER) in the serum specimens were detected by chemiluminescence immunoassay.Then the correlations between these markers were analyzed.Results All of the results showed that the expression of CXCR1, CXCR2 and CXCL8 at mRNA level in PBMCs from the PHC group were higher than those of the healthy control group (P<0.01) as well as those of the liver cirrhosis group (P<0.05).Up-regulated expression of CXCR1, CXCR2 and CXCL8 in patients with PHC were associated with the depth of tumor invasion, lymph node or distant metastasis, clinical stage and levels of CRP, AFP and FER in serum (P<0.05).The expression of CXCR1, CXCR2 and CXCL8 at protein level in liver biopsy tissues were also significantly increased in the PHC group in comparison with those of the healthy control group as indicated by the result of SP immunohistochemistry (P<0.05).Conclusion Levels of CXCR1, CXCR2 and CXCL8 in the patients with PHC are significantly increased and positively correlated with the levels of AFP, FER and CRP in serum, suggesting that the signal transduction process mediated by CXCR1, CXCR2 and their common ligand CXCL8 may play a key role in the pathological process of PHC.This study may provide a potential new strategy for immune intervention in hepatocellular cancer.

Expression of CXCL8 and its receptors (CXCR1 and CXCR2) in peripheral blood neutrophils of chronic hepatitis B / 中国免疫学杂志

Objective:To study on the levels of CXCL8 and its receptors (CXCR1 and CXCR2) in peripheral blood neutrophils of the patients with chronic hepatitis B.Methods:The neutrophils were isolated and purified by neutrophil isolation medium,and the loads of HBV-DNA in neutrophils were detected by PCR,and the levels of HBeAg in serum were measured by ELISA.The patients were divided into different groups according to the detective results so that the expressions of CXCL8 and its receptors ( CXCR1,CXCR2) in neutrophils were detected by the methods of streptavidin-biotin complex ( SABC ) immunocytochemistry stain.Results:The data of SABC immunocytochemical stain showed that the positive color of CXCL8 was mainly located in the cytoplasm of PMNs.However,the most positive color of CXCR1and CXCR2 was mainly expressed in the cytoplasm and cell membrane.Interestingly,the deeper immune coloring of CXCL8 and CXCR1, and relatively shallow immune coloring of CXCR2 were explored in the group with positive of HBeAg.The similar detective results also had been found in the cases with positive of HBV DNA in neutrophils.Compared with the normal control group,the levels of CXCL8 and CXCR1 in the patients were significantly increased ( P0.05).Conclusion:After neutrophils occult infected by HBV,not only the secretion of CXCL8 can be promoted, but also the expression of CXCR1 will be further increased.The data of immunohistochemical staining have been shown that the color degree of CXCL8 and its receptors ( CXCR1, CXCR2 ) are positive correlation to the level of HBeAg and the loads of HBV DNA.More PMNs can be chemotactic attraction to lesion so as to participate in the local inflammatory injury and tissue repair via the interactive pathway of the high expression of CXCR1 on surface of neutrophils with CXCL8.

Blockade of CXCR1/2 chemokine receptors protects against brain damage in ischemic stroke in mice

OBJECTIVE: Ischemic stroke may result from transient or permanent reductions of regional cerebral blood flow. Polymorphonuclear neutrophils have been described as the earliest inflammatory cells to arrive in ischemic tissue. CXCR1/2 receptors are involved in the recruitment of these cells. However, the contribution of these chemokine receptors during transient brain ischemia in mice remains poorly understood. In this work, we investigated the effects of reparixin, an allosteric antagonist of CXCR1/2 receptors, in a model of middle cerebral artery occlusion and reperfusion in mice. METHODS: C57BL/6J male mice treated with reparixin or vehicle were subjected to a middle cerebral artery occlusion procedure 1 h after the treatment. Ninety minutes after ischemia induction, the monofilament that prevented blood flow was removed. Twenty-four hours after the reperfusion procedure, behavioral changes, including motor signs, were analyzed with the SmithKline/Harwell/lmperial College/Royal Hospital/Phenotype Assessment (SHIRPA) battery. The animals were sacrificed, and brain tissue was removed for histological and biochemical analyses. Histological sections were stained with hematoxylin and eosin, neutrophil infiltration was estimated by myeloperoxidase activity and the inflammatory cytokine IL-iβ was measured by ELISA. RESULTS: Pre-treatment with reparixin reduced the motor deficits observed in this model of ischemia and reperfusion. Myeloperoxidase activity and IL-iβ were reduced in the reparixin-treated group. Histological analysis revealed that ischemic injury was also attenuated by reparixin pre-treatment. CONCLUSIONS: Our results suggest that the blockade of the CXCR1/2 receptors by reparixin promotes neuroprotective effects by reducing the levels of polymorphonuclear infiltration in the brain and the tissue damage associated with middle cerebral artery occlusion and reperfusion.

Study on CXCR1/CXCR2 antagonist G31P anti-inflammatory reaction mediated by neutrophils / 中华微生物学和免疫学杂志

Objective To Study on CXCR1/CXCR2 antagonist G31P anti-inflammatory reaction mediated by neutrophils.Methods Detect whether G31P can block chemotaxis of neutrophils induced by human IL-8 and inhibit the release of IL-8 by epithelia of segmental bronchus;establish HEK293 cell line transfected by pcDNA3.0-CXCR1 ,2,4 and detect the chemotaxis of IL-8 for HEK293 ;establish the experi-mental model of pneumonia induced by the P.aeruginosa,take count of the nucleated cells in the bronchoal-veolar lavage fluid(BALF),analyze myeloperoxidase(MPO) of lung tissue and observe the histopathology changing of it.Results G31P can inhibit the chemotaxis for neutrophils and transfected HEK293 cell line,inhibit the A549 releasing of inflammatory mediators;the proportion of neutrophils declines in G31P treat-ment group,pathology examination appears clear discrepancy.Conclusion G31P can block the chemotaxis of chemotactic factor with ELR+ CXC to neutrophils,block the combination of chemotactic factor with its re-ceptor CXCR2,block the CXCR2 on the surface of alveolar epithelia and vascular endothelial cells.Accordingly,neutrophils recruiting to topoinflammation can be prevented.

A novel mutation (Arg192Gly) in CXCR-1 gene detected in ankylosing spondylitis / 中华风湿病学杂志

Objective To search for the genetic and molecular immunity basis of CXCR-1 associated pathogenesis in ankylosing spondylitis (AS) patients. Methods Sequencing analysis was used to detect mutation in the exonic, junctional and promoter sequences of CXCR-1 which might be related with ankylosing spondylitis; the hydrophobicity, conservation and evolutionary distance of the mutated amino acids were also analyzed. Results Six affected individuals in the family were detected with a novel mutation Arg192Gly. The glycine at 192 codon was highly conserved in different species. Arginine and glycine had quite distinct hydrophobicity and BLOSUM score. Conclusion The mutation CXCR-1 (Arg192Gly) detected in these patients might be involved in genetic and molecular immunity mechnisms of ankylosing spondylitis.