Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system / 中国生物制品学杂志

@#Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.

Regulatory Mechanism of Mangiferin Combined with Bortezomib on Malignant Biological Behavior of Burkitt Lymphoma and Its Effect on Expression of CXC Chemokine Receptors / 中国实验血液学杂志

OBJECTIVE@#To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma.@*METHODS@#Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR).@*RESULTS@#Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05).@*CONCLUSION@#Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.

Scientific basis of acupuncture on mesenchymal stem cells for treating ischemic stroke / 中国针灸

The scientific basis of acupuncture on mesenchymal stem cells (MSCs) for treating ischemic stroke (IS) is discussed. MSCs transplantation has great potential for the treatment of tissue damage caused by early stage inflammatory cascade reactions of IS, but its actual transformation is limited by various factors. How to improve the homing efficiency of MSCs is the primary issue to enhance its efficacy. As such, the possible mechanisms of acupuncture and MSCs transplantation in inhibiting inflammatory cascade reactions induced by IS are explored by reviewing literature, and a hypothesis that acupuncture could promote the secretion of stromal cell-derived factor-1α (SDF-1α) from ischemic foci to regulate SDF-1α/CXC chemokine receptor 4 (CXCR4) axis, thereby improving the homing efficiency of MSCs transplantation, exerting its neuroprotective function, and improving the bed transformation ability, is proposed.

The Effects and Regulatory Mechanism of Targeting CXC Chemokine Receptor 1/2 Combined with Ara-C on the Malignant Biological Behaviors of U937 Cells of Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML.@*METHODS@#Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR).@*RESULTS@#Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05).@*CONCLUSION@#Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.

Effects of atractylodin on inflammatory damage of periodontal tissue and alveolar bone loss in periodontitis rats and its mechanism / 中国药房

OBJECTIVE To investigate the effects and mechanism of atractylodin on inflammatory injury of periodontal tissue and alveolar bone loss in periodontitis rats. METHODS A total of 144 SD rats were divided into control group （intragastric and intraperitoneal injection of normal saline）， model group （intragastric and intraperitoneal injection of normal saline）， atractylodin low-dose， medium-dose and high-dose groups （intraperitoneal injection of 6.665， 13.33， and 26.66 mg/kg atractylodin）， metronidazole group （positive control group， intragastric injection of 0.05 g/kg metronidazole， intraperitoneal injection of normal saline）， AMD3100 [stromal cell-derived factor-1 （SDF-1）/CXC chemokine receptor-4 （CXCR4） pathway inhibitor] group （intragastric injection of 1 mg/kg AMD3100， intraperitoneal injection of normal saline）， atractylodin high-dose+AMD 3100 group （intraperitoneal injection of 26.66 mg/kg atractylodin， intragastric injection of 1 mg/kg AMD3100）， with 18 rats in each group. Except for the control group， all other groups of rats were inoculated with Porphyromonas gingivalis to construct a periodontitis model. After successful modeling， they were given relevant medicine or normal saline， once a day， for 4 consecutive weeks. The gingival index of rats was detected； the levels of interleukin-6 （IL-6） and tumor necrosis factor α （TNF-α） in rat serum were also determined； alveolar bone resorption， periodontal histopathologic changes and the number of osteoclasts were detected by methylene blue staining， HE staining and TRAP staining， respectively. The expressions of osteoprotegerin （OPG）， receptor activator of NF-κB ligand （RANKL）， SDF-1 and CXCR4 proteins were determined. RESULTS Compared with the control group， serious pathological injury of periodontal tissue was found in the model group， the gingival index， the levels of IL-6 and TNF- α， alveolar bone absorption value， the number of osteoclasts， and the expression of RANKL protein were all increased significantly （P＜0.05）， while the expressions of OPG， SDF-1 and CXCR4 proteins were decreased significantly （P＜0.05）. Compared with the model group， pathological injury of periodontal tissue in rats was reduced； the gingival index， the levels of IL-6 and TNF-α， alveolar bone resorption value， osteoclast number and RANKL protein expression were decreased significantly， while protein expressions of OPG， SDF-1 and CXCR4 were increased significantly in atractylodin low-dose， medium-dose and high-dose groups and metronidazole group （P＜0.05）. The change trend of corresponding indexes in the AMD3100 group was opposite to the above （P＜0.05）. AMD3100 attenuated the inhibitory effect of high-dose atractylodin on inflammatory response and alveolar bone loss in rats with periodontitis （P＜0.05）. CONCLUSIONS Atractylodin may improve the inflammatory response and alveolar bone loss in periodontitis rats by activating the SDF-1/CXCR4 signaling pathway.

Epidemiological analysis on the susceptibility of CXCR3 gene polymorphism to COPD in the elderly population in Liangjiang New Area of Chongqing / 公共卫生与预防医学

Objective To explore and analyze the epidemiology of susceptibility to chronic obstructive pulmonary disease (COPD) among the elderly population in Liangjiang New Area of Chongqing based on CXC chemokine receptor 3 (CXCR3) gene polymorphism. Methods From January 2020 to September 2022, the Medical Laboratory Department of Chongqing Liangjiang New Area People's Hospital selected COPD patients and received treatment. Among the 276 patients who met the criteria were included in the study and included in the observation group. Among the 512 patients with healthy pulmonary function in the same period were included in the control group. The data of the two groups of patients were analyzed, and the genotypes were detected by SBaPhotoshot technology to analyze the relationship between gene polymorphism and the susceptibility and clinical characteristics of COPD. Results There was no significant difference between the two groups in age, sex, BMI and blood eosinophil granulocyte levels, which was comparable (P>0.05). There were significant differences in smoking history, pulmonary function index , MMP-9 and TIMP-1 levels (P0.05). In the observation group, the MMP-9 level of rs2280964 locus was significantly different (P=0.003), while the TIMP-1 level was not significantly different (P=0.187); There was no significant difference in MMP-9 and TIMP-1 levels among the three genes at rs34334103 locus (all P>0.05). The level of MMP-9 in homozygous TT patients with rs2280964 locus was significantly higher than that in homozygous CC patients (P=0.024). There were differences in FEV1/FVC of patients with CXCR3 rs34,334,103 gene distribution (P=0.008), among which there were significant differences in CC+CT and TT recessive models (P0.05). Conclusion CXCR3 gene polymorphism is significantly associated with the susceptibility to COPD, and also with the serum levels of MMP-9 and FEV1/FVC, which can be used as a new target for clinical research and treatment.

XCR1 Forms a Heterodimer with CXCR4 and Regulates Its Internalization / 中国生物化学与分子生物学报

Chemokine signal pathways are important for the regulation of tumour metastasis. Chemokine receptors CXCR4 (C-X-C chemokine receptor type 4) and XCR1 (chemokine XC receptor 1) are involved in the metastasis of breast cancer, while the interaction between them remains unclear. Here we first identified the interaction between CXCR4 and XCR1 based on membrane protein yeast two-hybrid assays. Bioluminescence resonance energy transfer (BRET) showed that XCR1 could competitively bind to CXCR4 to form a heterodimer (P < 0.01). Results of wound healing assays via transient transfection of XCR1 and CXCR4 into HEK293 cells showed that 41.55% of the migration area rate in the co-transformation group was lower than 58.75% in the CXCR4-alone group after adding 30 nmol/L S D F-β. The co-expression of XCR1 inhibited the cellular motility, possibly mediated by the SDF-1β (stromal cell-derived factor 1)/CXCR4 signal pathway (P < 0.05). Furthermore, CXCR4 on the cell surface after co-expression of XCR1 in CXCR4-EGFP transgenic HEK293 cells was detected by flow cytometry. And the result suggested that XCR1 could accelerate the internalization of CXCR4 into the heterodimer induced by 30 nmol/L SDF-1β (P<0.05), which increased the internalization rate from 14.38% to 64.10%. Finally, the phosphorylation of Akt and ERK, which were involved in cell proliferation and migration, respectively, were examined. After 10 minutes of SDF-1β stimulation, ERK phosphorylation in the CXCR4-alone group showed a 3.59-fold increase, whereas the increase of ERK phosphorylation in the co-transfected group was only 2.08-fold. Interestingly, heterodimer formation reduced the phosphorylation level of ERK and shortened the activation time, whereas the phosphorylation level of Akt remained unchanged. Collectively, our findings revealed the hetero-dimerization of CXCR4 and XCR1 and its effects on CXCR4-mediated cellular motility, receptor internalization, and ERK pathway phosphorylation. Therefore, XCR1-targeting drugs could be candidates for cross-desensitization of CXCR4 and might represent a possible option for inhibiting breast cancer metastasis.

CXCL8 Promotes M2 Macrophage Chemotaxis and Infiltration in Colorectal Cancer Microenvironment / 中国生物化学与分子生物学报

CXC chemokine ligand 8 (CXCL8) is highly expressed in many human tumors including colorectal cancer, and it can promote the malignant progression of tumors. It was reported that M2 macrophages were abundant in colorectal cancer microenvironment, but whether CXCL8 affects the infiltration of M2 macrophages and its potential mechanism are not yet clear. The study aimed to investigate the effect of CXCL8 on M2 macrophage infiltration and chemotaxis in the colorectal cancer. Firstly, we analyzed the CXCL8 expression and immune cell infiltration in human colorectal cancer tissues from TCGA RNA-seq data. The expression of CXCL8 was verified by immunohistochemistry in tissues obtained from Shanxi Provincial Cancer Hospital. Then, Western blot and qRT-PCR were employed to detect CXCL8 expression in five colorectal cancer cell lines. THP-1 cells were allowed to differentiate into M2 macrophages via the phorbol myristate acetate (PMA) and IL-4 treatment, followed by detection of the chemotaxis of M2 macrophages towards HCT116, SW480 and CXCL8-HCT116, CXCL8-SW480 cell lines. HCT116 and SW480 cells were treated with interleukin 1β (IL-1β) to detect the expression of CXCL8, and co-cultured with M2 macrophages to analyze the chemotactic activity. The results revealed that the expression of CXCL8 was increased in pairs of CRC tissues versus normal adjacent tissues, and there were more M2 macrophage infiltration in cancer tissues with high expression of CXCL8. The mRNA and protein expression of CXCL8 in HCT116 and SW480 were increased after the IL-1β treatment (P < 0. 05). We confirmed that CXCL8 is a chemotactic factor for M2 macrophages by transwell assays (P<0. 05). In conclusion, CXCL8 in colorectal cancer cells can be induced by IL-1β in colorectal cancer cells and the upregulation of CXCL8 can promote the chemotaxis of M2 macrophages. The massive infiltration of M2 macrophages in colorectal cancer microenvironment may be related with the increased expression of CXCL8.

Biological Functions of CXCL12-CXCR4 in Human Malignant Tumors / 中国生物化学与分子生物学报

Metastasis and cell infiltration are the difficulties in the treatment of solid and lymphatic carcinoma and the main causes of disease recurrence and death. The migration of cancer cells is a prerequisite for tumor metastasis and invasion. The CXCL12-CXCR4 pathway plays an important role in the pathogenesis of solid tumors and leukemia. The interaction between CXCL12 and its receptor CXCR4 can activate multiple signaling pathways and regulate different physiological and pathophysiological processes. Thus, blocking CXCL12-CXCR4 binding and / or downstream pathways has clinical benefits in treating a variety of diseases and cancers. Although some CXCL12 and CXCR4 antagonists have been identified and have shown encouraging results in terms of antitumor activity, these drugs have not been widely used in clinical patients due to their serious toxic and side effects. There is an urgent need to develop novel CXCL12-CXCR4 axis antagonists for the treatment of tumors. Herein, we review the recent research progress of CXCR4 pathway in solid tumors and leukemia, and discuss the therapeutic value and future research direction of CXCR4 pathways in solid tumors and leukemia.

Tumor microenvironment responsive liposomes blocking CXCL12/CXCR4 pathway and synergistically enhancing immune efficacy of anti-PD-L1 / 药学学报

Blocking immune checkpoint programmed cell death receptor 1 (PD-1) or programmed death receptor-ligand 1 (PD-L1) can enhance anti-tumor activity of effector T cells. However, the lack of response in many patients to PD-1/PD-L1 therapy remains a question. Improving the immunosuppressive tumor microenvironment (TME) to enhance the efficacy of immune checkpoint inhibitors has become a promising cancer treatment strategy. We constructed a liposome system (PD-L1/siCXCL12-Lp) of CXCL12 siRNA and anti-PD-L1 peptide with matrix metalloproteinases (MMPs) responsiveness, which combined the TME regulation of siCXCL12 and the immune regulation of anti-PD-L1 peptide. All animal experiments were approved by the Biomedical Ethics Committee of Peking University. The authors found that PD-L1/siCXCL12-Lp directly down-regulated the expression of CXCL12 in vitro (33.8%) and in vivo (15.5%). It also effectively increased the ratio of CD8+/Treg by 20.0%, which helped the anti-PD-L1 peptide to better exert its immune effect. The combination therapy significantly inhibited tumor growth (52.08%) with great safety, which explored a new idea for cancer immunotherapy.

Effect of CXC chemokine receptor 7 on cell cycle of neuronal cells in ischemic stroke / 中华危重病急救医学

Objective:To investigate the function and mechanism of CXC chemokine receptor 7 (CXCR7) in neuronal cells of ischemic stroke.Methods:The expression of CXCR7 in human neuroblastoma SH-SY5Y cells was interfered by small interfering RNA (si-RNA) technique. Oxygen-glucose deprivation/reoxygenation (OGD/R) injury model was constructed in SH-SY5Y cells. CXCR7 protein expression and cell cycle were detected by flow cytometry (FCM). The protein expression of CXCR7 and Akt signaling pathway was detected by Western blotting.Results:After 6 hours of OGD/R, the expression of CXCR7 was significantly decreased compared with OGD/R 0 hour (CXCR7/GAPDH: 0.483±0.098 vs. 1.000±0.000 by Western blotting and 0.686±0.0524 vs. 1.000±0.000 by FCM, both P < 0.01), cell cycle arrest in G0/G1 phase (1.190±0.040 vs. 1.000±0.000, P < 0.01). After CXCR7 si-RNA interference with SH-SY5Y cells, OGD/R was constructed again for 6 hours. Compared with negative control group (si-NC group) under the same environment, the expression of CXCR7 and phosphorylated Akt (p-Akt) was significantly decreased (CXCR7/GAPDH: 0.471±0.051 vs. 1.000±0.000, p-Akt/GAPDH: 0.616±0.027 vs. 1.000±0.000, both P < 0.001) and cell cycle arrest in G0/G1 phase (1.105±0.033 vs. 1.000±0.000, P < 0.05). Conclusion:The CXCR7 could regulate the cycle of neuronal cells in ischemic stroke through Akt signaling pathway, which has a protective effect on neuronal cells.

Research progress of C-X-C motif chemokine ligand 12 and its receptor signaling axis in the regulation of pulmonary fibrosis / 中华劳动卫生职业病杂志

Pulmonary fibrosis is an irreversible interstitial lung disease characterized by lung parenchyma remodeling and collagen deposition. In recent years, the incidence and mortality of pulmonary fibrosis caused by unknown causes have risen. However, its pathogenesis is still unclear. C-X-C motif chemokine ligand 12 (CXCL12)/C-X-C chemokine receptor 4 (CXCR4)/CXCR7 signal axis plays a critical regulatory role in pulmonary fibrosis disease. In addition, the signal axis has been shown to regulate recruitment and migration of circulating fibrocytes, mesenchymal stem cells to the damage lung tissue, the migration of endothelial cells, the proliferation and differentiation of fibroblasts and endothelial cells, which further affects the occurrence and progression of pulmonary fibrosis. In this review, we summarized the pathogenesis and treatment research progress of CXCL12 and its receptor CXCR4/CXCR7 in the occurrence and progression of pulmonary fibrosis.

CXCL5 inhibits tumor immune of lung cancer via modulating PD1/PD-L1 signaling / 中华肿瘤杂志

Objective: To investigate the role of CXCL5 in tumor immune of lung cancer and to explore the potential molecular mechanisms. Methods: A total of 62 cases of patients with lung cancer admitted in the First Affiliated Hospital of Henan University from May 2018 to December 2019 were recruited as study object. Another 20 cases of patients with pulmonary infectious diseases and 20 cases of healthy control were selected as control. Enzyme-linked immunosorbent assay (ELISA) was used to determine serum levels of CXCL5 in patients with lung cancer, pulmonary infectious diseases and healthy control. Immunohistochemical staining (IHC) was used to detect the expressions of CXCL5 and PD-1/PD-L1 in tumor and paracarcinoma tissues of patients with lung cancer. Pearson correlation analysis was used to evaluate the correlation between CXCL5 and PD-1 in tumor and paracarcinoma tissues of patients with lung cancer. Lewis cells either expressing CXCL5 or vector plasmids were used to establish C57BL/6J mice model of lung cancer, and all mice were then divided into vehicle and PD-1 antibody treatment groups, 10 mice for each group. The mice survival and tumor growth curves were recorded. IHC was used to evaluate the expressions of CXCL5, PD-1 as well as the proportions of CD8(+) T and Treg cells in xenograft tumor tissues. Results: In patients with lung cancer, the serum level of CXCL5 [(351.7±51.5) ng/L] was significant higher than that in patients with pulmonary infectious diseases and healthy control [(124.7±23.4) ng/L, P<0.001]. The expression levels of CXCL5 (0.136±0.034), CXCR2 (0.255±0.050), PD-1 (0.054±0.012) and PD-L1 (0.350±0.084) in tumor were significant higher than those in paracarcinoma normal tissues [(0.074±0.022), (0.112±0.023), (0.041±0.007) and (0.270±0.043) respectively, P<0.001]. CXCL5 was significant positively correlated with PD-1 in tumor tissues of lung cancer (r=0.643, P<0.001), but not correlated with PD-1 in paracarcinoma tissues(r=0.088, P=0.496). The vector control group, CXCL5 overexpression group, vector control + anti-PD-1 antibody treatment group and CXCL5 overexpression + anti-PD-1 antibody treatment group all successfully formed tumors in mice, while CXCL5 overexpression increased the tumor growth significantly (P<0.01), which was abrogated by the treatment of anti-PD-1 antibody. CXCL5 overexpression decreased the mice survival time significantly (P<0.01), this effect was also abrogated by the treatment of anti-PD-1 antibody. The proportion of CD8(+) T cells in CXCL5 overexpression group [(10.40±2.00)%] was significant lower than that in vector control group [(21.20±3.30)%, P=0.002]. The proportion of CD4(+) Foxp3(+) Treg cells in CXCL5 overexpression group [(38.40±3.70)%] was significant higher than that in vector control group [(23.30±2.25)%, P<0.001]. After the treatment of anti-PD-1 antibody, no significant difference were observed for the proportion of CD8(+) T cells [(34.10±5.00)% and (33.40±4.00)% respectively] and Treg cells [(14.70±3.50)% and (14.50±3.30)% respectively] in xenograft tumor tissues between CXCL5 overexpression+ anti-PD-1 antibody treatment group and vector control + anti-PD-1 antibody treatment group (P>0.05). Conclusion: The expressions of CXCL5 and PD-1/PD-L1 are all increased significantly in the tumor tissues of patients with lung cancer, CXCL5 may inhibit tumor immune of lung cancer via modulating PD-1/PD-L1 signaling.

The role and mechanism of CXCR4 in renal injury and fibrosis caused by calcium oxalate crystals / 中华泌尿外科杂志

Objective:To investigate the role and mechanism of chemokine receptor type 4 (CXCR4) in renal injury and fibrosis caused by calcium oxalate crystals in mice.Methods:In June 2021, Fifteen male C57/BL6 mice were divided into control group (5 mice), model group (5 mice), and AMD3100 intervention group (5 mice) by random number table method. In model group and AMD3100 intervention group, glyoxylate (100 mg/kg) was injected intraperitoneal for 7 consecutive days for modeling. Meanwhile, the AMD3100 intervention group was also given intraperitoneal injection of AMD3100 (1 mg/kg) for 7 days. The control group was continuously injected with equal volume saline intraperitoneally. After 7 days, peripheral blood was collected from each group to determine the levels of serum urea nitrogen (BUN) and creatinine (Scr) to assess the renal function; HE, Von-Kossa, Picrosirius Red staining was also taken from the left kidney to observe the pathological changes of renal tissue. CXCR4, transforming growth factor β1 (TGF-β1) were detected by immunohistochemistry and western blot. The expression levels of PI3K/AKT pathway-related proteins were detected by western blot.Results:The results of biochemical indexes showed that the serum Scr [(108.03±13.56) μmol/L vs. (39.50±4.48)μmol/L, P<0.01)] and BUN[(5.66±0.48)mmol/L vs. (0.77±0.10)mmol/L, P<0.01) levels were significantly increased in model group compared with the control group. The AMD3100 intervention group was significantly lower than the model group in terms of Scr [(65.77±3.27)μmol/L vs. (108.03±13.56)μmol/L, P<0.05) and BUN [(2.97±0.44)mmol/L vs. (5.66±0.48)mmol/L, P<0.05) levels. The results of kidney pathology in mice showed that renal tubules were significantly dilated with inflammatory cell infiltration in the model group compared with the control group, and a large number of calcium oxalate crystals and collagen fibers were deposited. The extent of kidney damage, calcium oxalate crystals and collagen fibers deposition were significantly reduced in the AMD3100 intervention group compared with the model group. The results of western blotting showed that the relative expression of CXCR4(0.639±0.019 vs. 0.158±0.012, P<0.01) and TGF-β1(0.698+ 0.018 vs. 0.314+ 0.015, P<0.05) was significantly increased in the model group compared with the control group. The relative expression of CXCR4(0.322±0.231) in the AMD3100 intervention group compared with the model group (0.322±0.231 vs. 0.639±0.019, P<0.05) and TGF-β1(0.445+ 0.017 vs. 0.698+ 0.018, P<0.05) were significantly decreased. The results of immunohistochemical staining showed the trend of CXCR4 and TGF-β1 expression in each group consistent with the results of protein blotting assay. Western blotting results showed that the expression of p-PI3K (0.613±0.016 vs. 0.213±0.011, P<0.01) and p-AKT(0.149±0.013 vs. 0.047±0.014, P<0.01) was significantly increased in the model group compared with the control group. The expression of p-PI3K in the AMD3100 intervention group compared with the model group (0.292±0.020 vs. 0.613±0.016, P<0.05) and p-AKT (0.098±0.021 vs. 0.149±0.013, P<0.05)was significantly decreased. Conclusion:CXCR4 inhibits calcium oxalate crystal-induced kidney injury and interstitial fibrosis in mice by targeting the PI3K/AKT pathway.

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.

Effect and Mechanism of Cxcr4 Gene-Modified BMSC-Derived Exosomes on Aplastic Anemia / 中国实验血液学杂志

OBJECTIVE@#To explore the improvement effect of CXC chemokine receptor 4 (Cxcr4) gene-modified bone marrow mesenchymal stem cell (BMSC)-derived exosomes on aplastic anemia (AA), and make a preliminary exploration of the mechanism.@*METHODS@#Mouse BMSCs were isolated and cultured, then infected by recombinant lentivirus carrying Cxcr4 gene. The expression of green fluorescence was observed through fluorescence microscope, the expression of Cxcr4 mRNA was detected by real-time fluorescence quantitative PCR, and the BMSC-derived exosomes modified with Cxcr4 gene were extracted. Mouse models of AA were constructed, and control group, model group (AA), AA+BMSC group, AA+NC-BMSC group, AA+Cxcr4-BMSC group were set up. Except control group and model group, the other three groups of mice were injected 400 μl exosomes from different sources via the tail vein, after 2 weeks, the routine blood indices and the number of bone marrow nucleated cells were detected, the pathological changes of bone marrow were observed by HE staining, and the expression level of Treg cells was detected by flow cytometry.@*RESULTS@#Mouse BMSCs were successfully isolated, and BMSCs with high expression of Cxcr4 and their exosomes were obtained. Compared with the control group, the number of red blood cell (RBC), white blood cell (WBC), and platelet (PLT), the hemoglobin (Hb) content and proportion of Treg cells in the peripheral blood of mice in the model group significantly decreased (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01). The proliferation level of nucleated cells was low, and the medullary cavity was filled with a large number of fat cells. Compared with the model group, the number of RBC, WBC, PLT, the Hb content and proportion of Treg cells in the peripheral blood of mice in the AA+BMSC group, AA+NC-BMSC group, and AA+Cxcr4-BMSC group significantly increased (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01), and pathological changes of bone marrow were improved. In addition, the number of RBC, WBC, PLT, the Hb content and proportion of Treg cells in the peripheral blood of mice in the AA+Cxcr4-BMSC group were significantly higher than those in the AA+BMSC group (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01).@*CONCLUSION@#Injection of Cxcr4 gene-modified BMSC-derived exosomes has a certain improvement effect on AA mice, and the mechanism may be related to an increase of the proportion of Treg cells.

Expression of CXC chemokine ligand 11 in gallbladder cancer and its effect on cell proliferation and invasion / 中华肝胆外科杂志

Objective:To investigate the expression of CXC chemokine ligand 11 (CXCL11) in gallbladder cancer (GBC) and its effect on cell proliferation and invasion.Methods:The surgically resected specimens of 47 GBC patients were collected in Lihuili Hospital Affiliated to Ningbo University from January 2017 to December 2020. There were 26 females and 21 males, with the age (62.0±8.2) years. The expression of CXCL11 protein in GBC tissues and corresponding paracancer tissues was detected by immunohistochemistry. Associations between CXCL11 expression and clinicopathological features were analyzed. After co-culturing of GBC-SD cells with exogenous CXCL11, cell counting kit-8 (CCK-8) and Transwell assays were performed to detect cell proliferation and invasion ability. The expression and phosphorylation level of phosphatidylinositol 3 kinase (PI3K) and protein kinase B (Akt) were also detected by Western blot.Results:The positive expression rate of CXCL11 in GBC tissues was significantly higher than that in adjacent paracancerous tissues [63.8% (30/47) vs 31.9% (15/47), χ 2=9.59, P=0.002]. Furthermore, CXCL11 expression was significantly associated with tumor stage (χ 2=6.64, P=0.010) and lymph nodal metastasis (χ 2=7.86, P=0.005). CCK-8 assay revealed that the proliferation ability of GBC-SD cells in CXCL11-treated group significantly increased than that in the control group (absorbance value: 0.59±0.06 vs 0.32±0.04, t=9.64, P<0.001). Transwell assay showed that the cell invasion ability in CXCL11-treated group significantly increased than that in the control group [number of transmembrane cells: (133.4±12.3) cells vs (38.6±4.4) cells, t=16.21, P<0.001]. Western blot analysis showed that the relative expression levels of phosphorylated PI3K (p-PI3K) and phosphorylated Akt (p-Akt) in CXCL11-treated group (0.88±0.06 and 0.83±0.04) were significantly higher than those in the control group (0.17±0.04 and 0.23±0.06), and the differences were statistically significant ( t=18.54, P<0.001 and t=15.21, P<0.001). Conclusion:CXCL11 is highly expressed in GBC and closely related to tumor progression. CXCL11 can promote the proliferation and invasion of GBC cells via PI3K/Akt signaling pathway.

Exploring the effect of C-X-C motif chemokine ligand-13 on the proliferation and migration of mesenchymal stem cells based on network pharmacology / 解剖学报

Objective To explore the effect of C-X-C motif chemokine ligand-13 (CXCL-13) on the proliferation and migration of human bone marrow mesenchymal stem cells (BMSCs) by network pharmacology. Methods To predict that the targets of CXCL-13 on BMSCs by online database. Metascape was used to perform gene ontology (GO) of the targets and Kyoto encyclopedia of genes and genomes (KEGG) pathway was used to perform enrichment analysis. The protein interaction analysis was performed by STRING 11.0 database, and the protein module of core gene was screened by using the cytoHubba 0. 1 of Cytoscape 3. 8. We divided BMSCs into control group, CXCL-13 group and PI3K inhibitor group. MTT assay, flow cytometric analysis and Transwell cell migration assay were respectively used to detect the absorbance (A) value of BMSCs in each group, the apoptosis rate and the number of cell migration. The protein contents of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in BMSCs supernatant were determined by ELISA. Western blotting was used to detect the protein expression of Akt and phosphorylated Akt (p-Akt) of BMSCs in each group. Results It was predicted that 21 targets of CXCL-13 effect on BMSCs. There were 32 biological processes related to cell proliferation include stem cell proliferation, regulation of endothelial cell proliferation and positive regulation of smooth muscle cell proliferation. There were 22 biological processes related to cell migration include regulating cell migration, amebic cell migration and endothelial cell migration. There were 40 KEGG pathways including cancer pathway, PI3K-Akt signaling pathway and MAPK signaling pathway. The core proteins included tumor protein P53 (TP53), epidermal growth factor receptor (EGFR), heat shock protein 90 kD alpha class B member 1 (HSP90AB1), protein kinase Ca (PRKCA), estrogen receptor 2 (ESR2) and prostaglandin E receptor 4 (PTGER4). Compared with other groups, the absorbance (A) value and cell migration number of BMSCs in CXCL-13 group increased significantly (P< 0. 01, 71=15), and the apoptosis rate decreased significantly (P<0. 01, n= 15). However, absorbance value, apoptosis rate and migration number of BMSCs in PI3K inhibitor group were contrary to those in CXCL-13 group (P<0. 01, n= 15). Compared with the control group, the protein contents of EGF and VEGF in BMSCs of CXCL-13 group increased significantly (P<0. 01, n= 15), and the relative expression of Akt and p-Akt increased significantly (P<0. 01, n = 9). However, the protein content of EGF and VEGF, and the relative expression of Akt and p-Akt in PI3K inhibitor group were opposite. Conclusion Through activating PI3K-Akt pathway, CXCL-13 may promote BMSCs paracrine EGF and VEGF proteins, and improve proliferation and migration of BMSCs, as well as inhibit BMSCs apoptosis.

Correlation between the expression of RON protein and CXC chemokine receptor 4 protein and abiraterone resistance in patients with castration-resistant prostate cancer / 中国临床药理学与治疗学

AIM: To study the correlation between the expression of recepteur d'origine nantais (RON) protein and CXC chemokine motif receptor 4 (CXCR4) protein and abiraterone resistance in patients with castration-resistant prostate cancer (CRPC). METHODS: From January 2017 to February 2020, 127 patients with CRPC who were treated with abiraterone in our hospital were selected. According to the status of drug resistance, they were divided into observation group (n=32, patients with abiraterone resistance) and control group (n=95, patients in remission). Immunohistochemistry and Western blot analysis were used to compare the expression of RON and CXCR4 protein between the two groups. Logistic regression analysis was used to conduct single-factor and multi-factor analysis of RON, CXCR4 protein and drug resistance, and receiver operating characteristic curve (ROC) and area under ROC (AUC) were used to analyze the value of RON and CXCR4 protein in predicting drug resistance. In addition, RON and CXCR4 inhibitors were added to abiraterone-resistant cell lines. The effects of the two on the apoptosis indicators of abiraterone resistance[caspase-3, caspase-9, apoptosis rate] were observed. RESULTS: Immunohistochemistry showed that the positive expression rate of RON in the observation group (71.88%, 23/32) was higher than that of the control group (27.37%, 26/95). The positive expression rate of CXCR4 in the observation group (65.63%, 21/32) was higher than that of the control group (12.63%, 12/95). Western blot detection showed that the RON and CXCR4 proteins in the observation group were higher than those in the control group (P 4.11, the sensitivity was 84.37%, and the specificity was 61.05% (P 3.42, the sensitivity was 75.00%, and the specificity was 80.00% (P< 0.05). The AUC of RON+CXCR4 protein predicting abiraterone resistance was 0.884 (95%CI: 0.815-0.934), the sensitivity was 87.50%, and the specificity was 83.16% (P< 0.05). CONCLUSION: The expression of RON and CXCR4 protein in CRPC patients increases significantly, which is closely related to the resistance of Abiraterone in patients and is expected to become a marker for predicting drug resistance. Inhibiting the expression of RON and CXCR4 proteins can promote the apoptosis of CRPC abiraterone resistant cells.

Changes analysis of CXCL12/CXCR4 in peripheral blood of patients with systemic lupus erythematosus before and after treatment / 中华风湿病学杂志

Objective:To observe the pattern change of chemokine (C-X-C motif) ligand 12 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) in systemic lupus erythematosus (SLE) by comparing the condition of SLE patients between pre-treatment and post-treatment.Methods:Peripheral blood mononuclear cells were obtained from 15 healthy controls and 8 first visit SLE patients before and after treatment. The expression of CXCR4, CD3, and CD19 on lymphocytes were detected by flow cytometry. CXCL12 concentration in serum was measured by enzyme-linked immunosorbent assay. For statistical analysis, the independent sample t test was used for independent samples of normally distributed data, the paired sample t test was used for paired samples of normally distributed data, the Mann-Whitney U test was used for independent samples of nonnormal distribution data, and the Wilcoxon test was used for comparison of related samples of non-normal distribution data. In the correlation analysis, Pearson correlation coefficient was used for data conforming to the normal distribution, and Spearman correlation coefficient was used for data not conforming to the normal distribution. Results:① The percentage of CXCR4 + lymphocyte subsets was significantly increased after treatment (90±7)% when compared with that before treatment (75±17)% ( t=-2.440, P<0.05). ② The mean fluorescence intensity of CXCR4 + lymphocyte subsets was significantly decreased after treatment [119.00(92.93, 142.50)] compared with that before treatment [177.00(130.75, 280.25)] ( Z=-2.100, P<0.05). ③ The plasma concentration of CXCL12 was significantly decreased after treatment (540±243) pg/ml when compared with that before the treatment (1 146±570) pg/ml ( t=3.219, P<0.05). ④ Moreover, there were significant correlations between the trends of the above three indicators after treatment. Conclusion:The proportion, the mean fluorescence intensity of CXCR4 + lymphocyte in peripheal blood subsets and the concentration of CXCL12 in plasma of first visit SLE patients are significantly changed after treatment. That suggests CXCL12/CXCR4 is closely associated with SLE.