OBJECTIVES: To investigate the effect of Shenge powder (SGP) on myocardial fibrosis in rats with heart failure after myocardial infarction and its relation with lysyl oxidase like protein 2 (LOXL2)/transforming growth factor-β1 (TGF-β1)/IL-11 signaling pathway. METHODS: Seventy-two SPF male SD rats were divided into blank control group, model control group, SGP small dose group, SGP large dose group, positive control group, SGP large dose+LOXL2 activator group, with 12 rats in each group. Except for the blank control group, post-myocardial infarction heart failure was induced by coronary constriction. Corresponding treatments were given immediately after successful modeling, once a day for 4 weeks. Left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) in rats were detected by color Doppler ultrasound imaging. Levels of IL-1β and IL-6 in serum were analyzed by ELISA method. Myocardial collagen volume fraction (CVF) was evaluated by Masson staining. Expressions of collagen Ⅰ and α-smooth muscle actin (α-SMA) in myocardial tissue were detected by immunohistochemical staining. The mRNA expressions of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in myocardial tissue were detected by qRT-PCR. Expression of LOXL2, TGF-β1, and IL-11 proteins in myocardial tissue were detected by Western blotting. RESULTS: Compared with the blank control group, the LVFS and LVEF of the model control group decreased, the levels of serum IL-6 and IL-1β elevated, and the CVF value, the expressions of collagen Ⅰ and α-SMA in myocardial tissue, MMP-9 and TIMP-1 mRNA, and LOXL2, TGF-β1, IL-11 proteins increased (all P<0.05). Compared with the model control group, the LVFS and LVEF of SGP small dose group, SGP large dose group and positive control group increased, the levels of serum IL-6 and IL-1β decreased, and the CVF value, the expressions of collagen Ⅰ and α-SMA in myocardial tissue, MMP-9 and TIMP-1 mRNA, and LOXL2, TGF-β1, IL-11 proteins decreased (all P<0.05); while LOXL2 activator reversed the improvement effect of high-dose SGP on myocardial fibrosis in heart failure rats after myocardial infarction. CONCLUSIONS Shenge powder may inhibit myocardial fibrosis in heart failure rats after myocardial infarction by inhibiting the LOXL2/TGF-β1/IL-11 pathway.
Animals ; Male ; Rats, Sprague-Dawley ; Myocardial Infarction/complications* ; Transforming Growth Factor beta1/metabolism* ; Signal Transduction/drug effects* ; Drugs, Chinese Herbal/therapeutic use* ; Rats ; Heart Failure/pathology* ; Myocardium/metabolism* ; Fibrosis ; Amino Acid Oxidoreductases/metabolism* ; Interleukin-11/metabolism* ; Tissue Inhibitor of Metalloproteinase-1/metabolism* ; Matrix Metalloproteinase 9/metabolism*

To observe the expression changes of interleukin-11 (IL-11) and its receptor in mice with pulmonary fibrosis. 
 Methods: C57BL/6 mice were randomly divided into a control group and a bleomycin (BLM) group (6 mice per group). BLM was injected into mice to induce idiopathic pulmonary fibrosis, while 50 μL PBS was injected into the control rats. The lung tissue was collected 21 d later. HE staining was used to observe morphological changes in lung tissue. Real-time PCR was used to detect IL-11 and its receptor gene expression. Western blot and immunohistochemical staining were used to detect IL-11 receptor expression. ELISA was used to detect the content of serum IL-11 in mice. In addition, the gene and protein expression of IL-11 receptor in mouse fibroblasts were detected by real-time PCR and Western blot, respectively. 
 Results: HE staining showed a significant change in pulmonary fibrosis in mice 21 d after BLM injection. The IL-11 mRNA expression in lung and IL-11 level in serum were up-regulated. The gene and protein expression of IL-11 receptor in mice with pulmonary fibrosis were significantly increased. The results from the cell experiments showed that the gene and protein expression of IL-11 receptor in mouse fibroblasts were significantly increased by TGF-β1.
 Conclusion: The expression of IL-11 and its receptor are up-regulated in mice with pulmonary fibrosis induced by BLM, which might be an important mechanism for the development of pulmonary fibrosis.
Animals ; Bleomycin ; Gene Expression Regulation ; Idiopathic Pulmonary Fibrosis ; physiopathology ; Interleukin-11 ; genetics ; Lung ; metabolism ; Mice ; Mice, Inbred C57BL ; Rats

A DNA fragment encoding recombinant human interleukin 11 (hrIL-11) was obtained by PCR from previously constructed pET24a-hrIL-11 plasmid. Then pET21a-hrIL-11 expression vector was constructed routinely and transformed into BL-21(DE3). By the induction of Isopropyl-1-thio-beta-D-galactoside (IPTG), hrIL-11 protein was highly expressed at about 20% of the total bacterial proteins and was identified by Western blot. After purification with Ni-NTA affinity chromatography and refolding with renaturation buffer, the purity of the target hrIL-11 protein reached 95% and its biology activity was 1 x 10(6) IU/mg, determined by stimulating the proliferation of T1165, which facilitates further researches into effects of IL-11 on platelet proliferation and other function.
Escherichia coli ; metabolism ; Genetic Vectors ; genetics ; Humans ; Interleukin-11 ; genetics ; metabolism ; Recombinant Proteins ; genetics ; isolation & purification ; metabolism

OBJECTIVETo investigate whether signaling through Toll-like receptor-2 (TLR-2) can affect the expression of some cytokines in human gingival fibroblasts.

METHODSThe gingival fibroblasts were isolated and cultured in vivo, divided into blank control group, lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg) group and Escherichia coli (Ec) group. mRNA expression levels were measured by real-time polymerase chain reaction (PCR). The protein expression levels were detected by the enzyme linked immunosorbent assay (ELISA). The data was statistically analyzed by SPSS16.0 software package.

RESULTSLPS from Pg could stimulate the expression of interleukin (IL)-6 and leukemia inhibitory factor (LIF) mRNA and protein, which reached the peak (5.87 ± 0.83) at 10 h, and the expression level increased with the increase of the Pg concentration. IL-11 or oncostatin-M (OSM) mRNA expression was not affected by LPS. After treated with Pg for 48 h, the protein expression of IL-6 and LIF was up-regulated, (962 ± 57) ng/L and (47 ± 18) ng/L respectively.

CONCLUSIONSSignaling through TLR-2 controls the expression of cytokines of IL-6 family in human gingival fibroblasts.

Adolescent ; Adult ; Cells, Cultured ; Child ; Dose-Response Relationship, Drug ; Escherichia coli ; chemistry ; Fibroblasts ; cytology ; metabolism ; Gingiva ; cytology ; Humans ; Interleukin-11 ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Leukemia Inhibitory Factor ; genetics ; metabolism ; Lipopeptides ; pharmacology ; Lipopolysaccharides ; administration & dosage ; isolation & purification ; pharmacology ; Oncostatin M ; genetics ; metabolism ; Porphyromonas gingivalis ; chemistry ; RNA, Messenger ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; agonists ; Young Adult

OBJECTIVETo investigate the binding characteristics of interleukin 11 (IL-11) analogue-cyclic nonapeptide c(Cys-Gly-Arg-Arg-Ala-Gly-Gly-Ser-Cys) NH2 C30H54N16O10S2, c(CGRRAGGSC), and human prostate cancer PC-3 cells.

METHODSc(CGRRAGGSC) was labeled with fluorescent dye LSS670, and the location of LSS670-cyclic nonapeptide in the PC-3 cells was investigated by fluorescent microscopy. Flow cytometry was used to detect the fluorescence intensity of the in vitro binding of LSS670-c (CGRRAGGSC) to PC-3 cells and calculate its IC50 and Ki in competitive inhibition experiments. 99Tcm-DTPA-c(CGRRAGGSC) was synthesized by the reaction of 99mTcO4- with c(CGRRAGGSC). The binding characteristics of 99mTc-DTPA-c(CGRRAGGSC) and IL11R in the PC-3 cells were analyzed by radioreceptor assay. Bmax and Kd were calculated in saturability and reversibility experiments.

RESULTSThe binding of LSS670-c(CGRRAGGSC) to the PC-3 cells showed the characteristics of saturability and concentration-time dependence. Unlabeled c(CGRRAGGSC) and LSS670-c(CGRRAGGSC) exhibited a competitive inhibition on the PC-3 cells (IC50 = [6.31 +/- 0.12] nmol/L, Ki = [2.11 +/- 0.14] nmol/L). Fluorescence was mainly distributed in the cell membrane (Kd = [0.32 +/- 0.02] nmol/L, Bmax = [754 +/- 34] fmol/mg pro).

CONCLUSIONc (CGRRAGGSC) could bind PC-3 cells through a receptor-mediated pathway.

Cell Line, Tumor ; Humans ; Interleukin-11 ; metabolism ; Male ; Peptides ; metabolism ; Prostatic Neoplasms ; metabolism ; Protein Binding

OBJECTIVETo study the immunomodulatory effects and mechanisms of mesenchymal stem cells (MSC) derived from the bone marrow in acute leukemia patients in vitro.

METHODSBone marrow mononuclear cells from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) were obtained and cultured in low serum medium. The immunophenotypes were assessed by FACS and immunol histochemistry. The levels of cytokines were evaluated by enzyme linked immunosorbant assay (ELISA). T-cell suppression ability was evaluated by Transwell chamber assay. Moreover, the immunoregulatory ability of AML- and ALL-derived MSC was detected by mixed lymphocyte culture assay.

RESULTSALL-derived MSC showed a typical fibroblast-like morphology. They were positive for CD29, CD44 and CD105, the positive rate were 98.81%, 99.25% and 90.52%, respectively, while negative for CD31, CD45 and CD34. Moreover, ALL- and AML-derived MSC didn't express HLA-DR and co-stirnulatory molecules (CD40, CD80 and CD86). ALL and AML derived MSC could secret several cytokines, such as TGF-β1 (567.58 ± 52.64 and 357.15 ± 33.52), HGF (647.27 ± 102.54 and 219.67 ± 62.37), IL-6 (59.67 ± 15.69 and 54.35 ± 12.31) and IL-11 (102.58 ± 23.54 and 78.21 ± 9.67), the level of secretion of TGF-β1 and HGF were higher in ALL bone marrow derived MSC than that of in AML bone marrow derived MSC. ALL and AML derived MSC significantly suppressed T lymphocyte proliferation in a dose-dependent manner, the counts per minute (CPM) were (3.58 ± 0.54) × 10(4), (2.87 ± 0.33) × 10(4), (1.78 ± 0.51) × 10(4) and (1.15 ± 0.15) × 10(4) for AML derived MSC, and CPM were (1.96 ± 0.31) × 10(4), (1.57 ± 0.28) × 10(4), (0.91 ± 0.41) × 10(4) and (0.22 ± 0.11) × 10(4) for ALL derived MSC when MSC were 0.5 × 10(4), 1 × 10(4), 2 × 10(4) and 5 × 10(4). In addition, the CPM was (4.01 ± 0.72) × 10(4) in control group. The immunosuppressive ability was different between MSCs derived from AML and ALL. The immunosuppressive effect of ALL derived MSC could be reversed by anti-TGF-β1 and anti-HGF antibody.

CONCLUSIONALL-derived MSC show immunoregulatory effect in vitro and this effect is achieved through cytokines. But MSCs derived from AML display abnormal changes in T-cell suppression ability.

Bone Marrow ; immunology ; Bone Marrow Cells ; cytology ; Cell Proliferation ; drug effects ; Cytokines ; pharmacology ; Humans ; Immunophenotyping ; Interleukin-11 ; metabolism ; Interleukin-6 ; metabolism ; Leukemia, Myeloid, Acute ; immunology ; Mesenchymal Stromal Cells ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; Transforming Growth Factor beta1 ; metabolism

The aim of this study was to investigate the role of mesenchymal stem cells in the hematopoietic reconstitution of patients who had received haploidentical allogeneic hematopoietic stem cell transplantation (hi-allo-HSCT). 15 patients who underwent treatment with both MSCs and HSCs, were selected as study group, while 20 patients receiving only HSCT were taken as control. Bone marrow samples were obtained from iliac crest aspirates at several times after HSCT for the isolation, purification and expansion of MSCs. The confluent ratio and time were measured and compared with those of the control. The peripheral blood samples were obtained from patients, then absolute neutrophil and platelet counts were assayed. From day 4 before transplantation to day 28 after transplantation, serum was obtained every four days from patients of the two groups, and then 3 cytokines as SDF-1alpha, TPO and IL-11 were detected by ELISA. The results indicated that as compared with the control group, the ratio of primary confluent layer formation of MSCs in study group was obviously higher (27.3%) (p < 0.01), and the confluence time in culture was significantly less (p < 0.05). In the study group, the concentration of SDF-1alpha amounted to peak value (2975.19 +/- 681.56 pg/ml) on the 8th day after HSCT, which was obviously higher than that before HSCT (2403.70 +/- 522.39 pg/ml, p < 0.05), whereas in the control, the concentration of highest point of SDF-1alpha reached to peak valve (2280.60 +/- 701.25 pg/ml) on the 16th day after HSCT, which was less than that before HSCT (2701.46 +/- 483.21 pg/ml, p < 0.05). The concentration of TPO and IL-11 was higher in study group compared with the control from day 16 to 28 after HSCT (p < 0.05). It is concluded that the transfusion of MSCs combined with hi-all-HSCT may improve the injured state of the hematopoietic microenvironment in bone marrow of patients during allo-HSCT.
Adolescent ; Adult ; Bone Marrow ; metabolism ; pathology ; Chemokine CXCL12 ; metabolism ; Child ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic System ; Humans ; Interleukin-11 ; metabolism ; Mesenchymal Stem Cell Transplantation ; methods ; Middle Aged ; Thrombopoietin ; metabolism ; Young Adult

BACKGROUNDBone morphogenetic protein (BMP) is a member of the superfamily of transforming growth factor-beta. Recent studies show that it is an indispensable factor in hematopoiesis. To better characterize the effect of recombinant human BMP (rhBMP)-2 in hematopoiesis, we set out to determine whether rhBMP-2 could promote the proliferation of mesenchymal stem cells (MSCs) and increase the levels of hematopoietic cytokines in MSCs.

METHODS2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino) carbonyl)-2H-tetrazolium hydroxide (XTT), real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of rhBMP-2 on the proliferation and hematopoietic cytokine levels of MSCs. In addition, MSCs marked with Hoechst33342 were transplanted into BALB/c mice by the intravenous route or intra-bone marrow transplantation, and cluster numbers were counted.

RESULTSThe XTT test revealed that rhBMP-2 significantly induced proliferation of MSCs in doses ranging from 10 ng/ml to 0.1 mg/ml in a dose-dependent manner. The experiments in vivo showed that there were more clusters of donor cells in bone marrow, spleen, liver and lung of the BMP group than those in the control group after both intra-bone marrow transplantation (P < 0.001, P < 0.001, P < 0.001, and P = 0.001, respectively) and intravenous transplantation (P < 0.001, P < 0.001, and P < 0.001 respectively). The results of real-time PCR and ELISA revealed that rhBMP-2 significantly increased mRNA expressions and protein levels of IL-6, IL-7, IL-11, G-CSF, M-CSF and SCF.

CONCLUSIONSThe treatment with rhBMP-2 promotes the proliferation of MSCs in vivo and in vitro and increases the levels of hematopoietic cytokines in MSCs, which may contribute to the improvement of hematopoietic function.

Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Granulocyte Colony-Stimulating Factor ; genetics ; Humans ; Interleukin-11 ; genetics ; Interleukin-6 ; genetics ; Interleukin-7 ; genetics ; Macrophage Colony-Stimulating Factor ; genetics ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred BALB C ; Polymerase Chain Reaction ; Recombinant Proteins ; pharmacology ; Stem Cell Factor ; genetics ; Transforming Growth Factor beta ; pharmacology

The study was aimed to investigate the technological parameters of rhIL-11 preparation from fusion protein by using enterokinase. Fusion expression vector pET32a/IL-11 was transferred into E.coli BL21 (DE3) pLysS and its expression was induced by IPTG, the lysis supernatant of the engineering strain was purified by Ni-NTA resin and then the target rhIL-11 was digested by auto-cleavaged DsbA-EKL-(His)(8). The results showed that after affinity chromatography, Trx-IL-11 was obtained with the yield of 11.25mg/g, the purity of 89.2% and the recovery of 91.8%. Finally the target rhIL-11 was digested and purified to over 95%. In conclusion, the preparation method of rhIL-11 from fusion protein by using enterokinase is simple and feasible with good separation, which can meet industrial requirements.
Enteropeptidase ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Interleukin-11 ; biosynthesis ; genetics ; Protein Engineering ; methods ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo observe the effects of Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa), Escherichia coli (Ec) lipopolysaccharides (LPS) on the production of IL-11 and IL-6 from healthy human gingival fibroblasts (HGF), and the effects of endogenous prostaglandin on HGF IL-11 and IL-6 production stimulated with the above LPS.

METHODSHGF were stimulated with Pg-, Aa-, Ec-LPS of different concentrations (0.1, 1, 10 mg/L) for 24 h. And HGF were also stimulated with the combinations of 10 mg/L Pg-, Aa-, Ec-LPS and 10(-6) mol/L indomethacin respectively for 24 h. Levels of IL-11 and IL-6 in the supernatants were quantitated by ELISA.

RESULTSLPS from Aa, Ec, at the concentration of 10 mg/L and from Pg at the concentrations 1, 10 mg/L significantly augmented IL-11 production by HGF. IL-6 production was also significantly increased by stimulation with Aa-LPS at concentrations 1, 10 mg/L and with Ec-, Pg-LPS at concentrations 0.1, 1, 10 mg/L. In addition, IL-11 production was lower than IL-6 production by HGF stimulated with LPS. Indomethacin significantly inhibited IL-6 and IL-11 production in LPS-stimulated HGF.

CONCLUSIONSAa-, Pg-, Ec-LPS may significantly increase IL-11 and IL-6 level in the supernatants of HGF, and endogenous prostaglandin may upregulate IL-11 and IL-6 production in LPS-stimulated HGF.

Aggregatibacter actinomycetemcomitans ; chemistry ; Cells, Cultured ; Dose-Response Relationship, Drug ; Escherichia coli ; chemistry ; Fibroblasts ; drug effects ; metabolism ; Gingiva ; cytology ; metabolism ; Humans ; Indomethacin ; pharmacology ; Interleukin-11 ; biosynthesis ; Interleukin-6 ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Porphyromonas gingivalis ; chemistry