Objective To explore immunosuppressive effect and sappan L （WECSL） in heart transplantation of rats. Methods mechanism of watery extract of caesalppinia Wistar rats （donor） heart allografts were transplanted into the abdomen of SD rats （receptor）. Ninety-six SD rats were divided into four groups （24 rats in each group）. Control group： olive oil（8ml/kg·d） treated; group A： cyclosporine A （CsA,5ml/kg·d） treated; groupB： WECSL（37.5g/kg·d） treated; group C： WECSL（25g/kg·d） plus semidose of CsA（2.5mg/kg·d） treated. Median survival time of heart allografts and the histological changes of allografts were examined. Messenger ribonucleic acid （mRNA） of interleukin-2（IL-2）, interleukinf-10（IL-10） in the myocardium were determined by reverse transcription polymerase chain reaction （RT-PCR）, serum level of IL-2 and IL-10 were determined by enzyme-linked immunosorbent assay （ELISA） at 3, 7 days after surgery. Results Compared with control group, median survival time of heart allografts in group A, group B, group C was prolonged （P〈0. 01）, lymphocyte infiltration and myocyte necrosis were relieved, mRNA expression of IL-2 in allografts was lower, mRNA expression of IL-10 was higher （P〈0.01）. The serum levels of IL-2 in group A 3,7days after surgery and in group B, group C 3 days after surgery was lower than that in control group （P〈0.01）. The serum levels of IL-10 in group A 7days after surgery and in group B 3 days after surgery was higher than that in control group （P〈0. 05）. Conclusion Acute rejection of rat heart transplantation can be effectively suppressed by WECSL, Th1 to Th2 polarization induced by WECSL is observed.
Objective To investigate the feasibility of recombinant lentivirus (LVs) mediated hyperpolarization- activated cyclic nucleotide-gated cation channel 4 (HCN4) gene transfecting rat bone mesenchymal stem cells (BMSCs) so as to construct the biological pacemaker cells. Methods Sprague Dawley rats at the age of 3-5 weeks were selected to isolate and culture BMSCs using modified whole bone marrow adherent culture method. LVs was used as carrier, and enhanced green fluorescent protein (EGFP) as marker to build LVs-HCN4-EGFP virus liquid. The BMSCs at passage 3 were transfected with LVs-HCN4-EGFP virus liquid (experimental group) and LVs-EGFP null virus liquid (control group). Fluorescence microscope was used to observe the green fluorescent protein expression after 24, 48, and 72 hours of transfection; Western blot method was used to detect the HCN4 protein expression. The electrophysiology was used to detect the pacemaker current in the experimental group. Results After transfection, BMSCs in the experimental group showed normal morphology and good growth; scattered green fluorescence could be seen at 48 hours under fluorescence microscope, with a transfection efficiency of about 10%; the fluorescence expression increased slightly, with the transfection efficiency of 20% to 25% at 72 hours. While no expression of green fluorescence was seen in the control group. Western blot results showed that the same band expression as a relative molecular mass of HCN4 protein were found at 72 hours after transfection in the experimental group, only weak expression of protein band was seen in the control group; the gray value of the experimental group (33.75 ± 0.41) was significantly higher than that of the control group (23.39 ± 0.33) (t=17.524, P=0.013). In the experimental group, the pacemaker current was recorded, and it could be blocked by CsCl, in accordance with the characteristics of pacemaker current. Conclusion The recombinant LVs mediated HCN4 gene is successfully transfected into rat BMSCs, and the expression of HCN4 protein and the pacemaker current can be detected.
【Abstract】 Objective To investigate the expression of connexin 40 (Cx40) and hyperpolarization-activated cycl icnucleotide-gated cation channel 4 (HCN4) in rat bone marrow mesenchymal stem cells (BMSCs) cocultured with the sinoatrialnode (SAN) tissues in vitro, so as to evaluate the possibil ity of BMSCs differentiation into SAN cells. Methods BMSCs wereisolated from Sprague Dawley rats (aged 4-6 weeks, male or female) by the adhesive method and cultured; BMSCs at the 3rdpassage were marked with carboxyfluorescein succinimidyl ester, and then were incubated on 6-well culture plate; cell climingsl ices were prepared at the same time. SAN tissue was taken and cut into 0.3 cm × 0.3 cm mass, and then placed into 4℃ PBSsolution. The SAN tissue mass was cocultured with marked BMSCs at the 3rd passage for 3 weeks as the experimental group, andBMSCs at 3rd passage were cultured alone for 1 week as the control group. At 1, 2, and 3 weeks after coculture, the mean integratedabsorbance (MIA) values of Cx40 and HCN4 were measured by Image pro plus 5.0 through the method of immunohistochemistry,and the mRNA expressions of Cx40 and HCN4 were identified by real-time fluorescent quantitative PCR. Results TheMIA values of Cx40 and HCN4 in the experimental group were higher than that in the control group, showing significantdifferences (P lt; 0.01). In the experimental group, the expressions of Cx40 and HCN4 increased gradually with time. The longerthe culture time was, the higher the expressions of Cx40 and HCN4 were, showing significant differences (P lt; 0.05). The mRNAexpressions of Cx40 and HCN4 in the experimental group were significantly higher than those in the control group (P lt; 0.01); inthe experimental group, the mRNA expressions of Cx40 and HCN4 increased gradually with time, showing significant differencesbetween different time points (P lt; 0.05). Conclusion The expressions of Cx40 and HCN4 increase obviously after coculturingBMSCs with SAN tissue, indicating that BMSCs could differentiate into SAN cells by coculturing with SAN tissue in vitro.
Objective To study the influence of ischemia-reperfusion on the expression of the hyperpolarization activated cycl icnucleotide gated cation channel 4 (HCN4) and to discuss the mechanism of functional disturbance of sinoatrial node tissue (SANT) after ischemia reperfusion injury (IRI). Methods Eighty five healthy adult rabbits, weighing 2-3 kg, were randomly divided into 3 groups: control group [a suture passed under the root section of right coronary artery (RCA) without l igation, n=5], experimental group A (occluding the root section of RCA for 30 minutes, then loosening the root 2,4, 8 and 16 hours, n=10), experimental group B (occluding the root section of RCA for 1 hour, then loosening the root 2, 4,8 and 16 hours, n=10). At the end of the reperfusion, the SANT was cut off to do histopathological, transmission electronmicroscopical and immunohistochemical examinations and semi-quantitative analysis. Results The result of HE stainingshowed that patho-injure of sinoatrial node cell (SANC) happened in experimental groups A and B after 2 hours of reperfusion, the longer the reperfusion time was, the more serious patho-injure of SANC was after 4 and 8 hours of reperfusion, SANC reached peak of damage after 8 to 16 hours of reperfusion; patho-injure of SANC was more serious in experimental group B than in experimental group A at the same reperfusion time. Immunohistochemical staining showed that the expression of HCN4 located in cellular membrane and cytoplasm in the central area of SANC and gradually decreased from the center to borderl ine. The integral absorbance values of HCN4 expression in the control group (397.40 ± 34.11) was significantly higher than those in the experimental group A (306.20 ± 35.77, 216.60 ± 18.59, 155.40 ± 19.11 and 135.00 ± 12.30) and in the experimental group B (253.70 ± 35.66, 138.70 ± 13.28, 79.10 ± 9.60 and 69.20 ± 8.42) after 2, 4, 8 and 16 hours of reperfusion (P lt; 0.05). With reperfusion time, the expression of HCN4 of SANC decreased, which was lowest after 8 hours of reperfusion; showing significant difference among 2, 4 and 8 hours after reperfusion (P lt; 0.05) and no significant difference between 8 and 16 hours after reperfusion (P gt; 0.05). At the same reperfusion time, the expression of HCN4 was higher in the experimental group A than in the experimental group B. The result of transmission electron microscope showed that ultramicrostructure of SANC was damaged after reperfusion in experimental groups A and B. The longer the reperfusion time was, the more serious ultramicrostructure damage of SANC was, and reached the peak of damage after 8 hours of reperfusion. Ultramicrostructure of SANC was not different between 8 and 16 hours of reperfusion. At the same reperfusion time, the ultramicrostructure damage of SANC was moreserious in experimental group B than in experimental group A. Conclusion IRI is harmful to the morphous and structure ofSANC, and effects the expression of HCN4 of SANC, which is concerned with functional disturbance and arrhythmia.
In order to explore the application of the dielectric properties of white matter and grey matter in β, δ and γ dispersion transition zones used in clinical medicine and microwave imaging technology, we calculated the dielectric constant and its increment by using Cole-Cole equation. Based on the mutation of the increment of dielectric constant, the frequency range of three dispersions were evaluated. The dominate dispersion and the corresponding polarization mechanism were analyzed by using Cole-Cole circle. The results showed that there are 3 transition zones in brain white matter, which occur between β and δ dispersion, δ and γ dispersion and β and γ dispersion respectively. In grey matter, there are only 2 transition zones, which are between β and δ dispersion and δ and γ dispersion respectively. By comparing the frequency range of white matter and grey matter, the frequency range in white matter is broader than that in grey matter for the transition zone of β and δ dispersion with the β dispersion occupying dominate position in both tissues, and the corresponding polarization mechanism is interfacial polarization. For the transition zone of δ and γ dispersion, the frequency range in white matter is also broader than that in grey matter with the δ dispersion occupying dominate position in both tissues, and the corresponding polarization mechanism is orientation polarization. This study can provide basic theory and reference for diagnosis of brain diseases and microwave imaging technology.
ObjectiveTo lay a theoretical foundation for the research of regulation of Hyperpolarization activated cyclic nucleotide gated channel 1 (HCN1) gene expression and its involvement in the pathogenesis of Mesio-temporal lobe epilepsy (MTLE) and other related diseases, the bioinformatics methods were used to analyze sequence characteristic, transcription factors and their binding sites in the promoter region of human HCN1 gene, and the physicochemical properties, signal peptides, hydrophobicity, transmembrane regions, protein structure, interacting proteins and functions of HCN1 proteins.MethodBiological software and website, such as Protparam, Protscale, MHMM, SignalP 5.0, NetPhos 3.1, Swiss-Model, Promoter 2.0, AliBaba2.1 and EMBOSS were used to analyze and predict physicochemical properties, structural functions, localized expression, phylogenetic relationships and protein interactions with human HCN1 protein, and promoter, CpG island and transcription factor characteristics of HCN1 gene.ResultsThe evolutionary analysis of HCN1 protein showed that the genetic distance between human and Pongo abelii was the smallest, indicating the closest genetic relationship between human and Pongo abelii. Human HCN1 protein was an unstable hydrophilic protein located on the plasma membrane, which contained two transmembrane structure. However, the predicted results showed that there was no signal peptide and nuclear localization sequence in this protein. The secondary structure of HCN1 protein was mostly random coil and alpha helix, and it contained multiple potential phosphorylation sites. The ontology analysis results of HCN1 protein were showed as follows. The cellular component of HCN1 protein was located in the plasma membrane (GO:0005886); the molecular functionof HCN1 protein were cyclic adenosine monophosphate binding (GO:0030552) and voltage-gated ion channel activity (GO:0005244); the biological process of this protein were reacting to cAMP (GO:0071320) and transmembrane transport of potassium (GO:0071805). The analysis results of String database showed that the proteins that had close interaction with human HCN1 protein mainly included the ten proteins (HCN2, HCN4, PEX5L, MARCH7, KCTD3, GNAT3, SHKBP1, KCNQ2, FLNA and NEDD4L). These proteins were mainly involved in regulation of ion transport and transmembrane transport of potassium (GO:0071805). The HCN1 gene was located at 5p12 and contained 8 exons and 7 introns.There were at least three promoter regions in the nucleotide sequence of 2 000 bp from the upstream of the HCN1 gene to the 5 'flanks, and contained a 158 bp CpG island in the promoter region and one TATA boxes and one CAAT boxes in the 5' regulation region ofHCN1 gene; niceteen transcription factors, including NF-κB, NF-1, AP-1, TBP, IRF-1, c-Ets-1, Elf-1, HNF-3, HNF-1, YY1, GATA-1, RXR-α, GR, AP-2αA, ENKTF-1, C/EBPβ, C/EBPα, c-Fos and c-Jun, binding in the promoter region of the HCN1 gene were predicted by both softwares (AliBaba2.1 and PROMO2).ConclusionThe analysis results provide important information for further studies on the role of HCN1. Bioinformatics analysis of the promoter region can improve the research efficiency of gene promoters, and provide theoretical basis for subsequent experiments to construct expression vectors of HCN1 gene promoters and identify their functions.
ObjectiveTo construction the telmisartan/collagen/polycaprolactone (PCL) nerve conduit and assess its effect on repairing sciatic nerve defect in rats. Methods The 60% collagen/hexafluoroisopropanol (HFIP) solution and 40% PCL/HFIP solution were prepared and mixed (collagen/PCL solution). Then the 0, 5, 10, and 20 mg of telmisartan were mixed with the 10 mL collagen/PCL solution, respectively. Telmisartan/collagen/PCL nerve conduits were fabricated via high voltage electrospinning technology. The structure of nerve conduit before and after crosslinking was observed by using scanning electron microscope (SEM). The drug release efficiency was detected by in vitro sustained release method. RAW264.7 cells were cultured with lipopolysaccharide to induce inflammation, and then co-cultured with nerve conduits loaded with different concentrations of telmisartan for 24 hours. The mRNA expressions of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg-1) were detected by using real-time fluorescence quantitative PCR. Forty adult Wistar rats were randomly divided into 4 groups (n=10). After preparing 15-mm-long sciatic nerve defect, the defect was repaired by cross-linked nerve conduits loaded with 0, 5, 10, and 20 mg telmisartan in groups A, B, C, and D, respectively. After operation, the general condition of rats was observed after operation; the sciatic function index (SFI) was tested; the bridging between the nerve conduit and sciatic nerve, and the integrity of nerve conduit were observed; the tissue growth in nerve conduit and material degradation were observed by HE staining; the expressions of CD86 (M1 macrophage marker), CD206 (M2 macrophage marker), myelin basic protein (MBP), and myelin protein 0 (P0) in new tissues were also observed by immunohistochemical staining; the expressions of neurofilament 200 (NF-200) and S-100β in new tissues were assessed by immunofluorescence staining. Results The general observation showed that the inner diameter of the nerve conduit was 1.8 mm and the outer diameter was 2.0 mm. After cross-linking by genipin, the nanofiber became thicker and denser. The drug release test showed that the telmisartan loaded nerve conduit could be released gradually. With the increase of telmisartan content in nerve conduit, the iNOS mRNA expression decreased and the Arg-1 mRNA expression increased; and the differences between 20 mg group and other groups were significant (P<0.05). In vivo experiment showed that all animals in each group survived until the completion of the experiment. The SFI was significantly higher in groups C and D than in groups A and B at different time points (P<0.05) and in group D than in group C at 6 months after operation (P<0.05). HE staining showed that there were significantly more new tissues in the middle of the nerve conduit in group D after operation than in other groups. Immunohistochemical staining showed that CD86 and CD206 stainings were positive in each group at 1 month after operation, among which group D had the lowest positive rate of CD86 and the highest positive rate of CD206, and there were significant differences in the positive rate of CD206 between group D and groups A, B, and C (P<0.05); the MBP and P0 stainings were positive in groups C and D at 6 months, and the positive rate in group D was significantly higher than that in group C (P<0.05). Immunofluorescence staining showed that the NF-200 and S-100β expressions in group D were significantly higher than those in other groups. ConclusionTelmisartan/collagen/PLC nerve conduit can promote the sciatic nerve defect repair in rats through promoting the polarization of M1 macrophages to M2 macrophages, and the nerve conduit loaded with20 mg telmisartan has the most significant effect.
Objective To explore the differences in lung function, neutrophil polarization, and serum total immunoglobulin E (IgE) levels among bronchial asthma patients, chronic obstructive pulmonary disease (COPD) patients, and asthma-COPD overlap syndrome (ACO) patients. Methods The retrospective analysis enrolled 127 patients with respiratory system diseases diagnosed and treated in Wuwei People’s Hospital between March 2016 and March 2019. Among them, 45 patients with moderate and severe bronchial asthma were in included the asthma group, 42 patients with acute exacerbations of COPD were included in the COPD group, and 40 patients with moderately persistent and severely persistent ACO were included in the ACO group. Forty-eight healthy examinees in the same period were selected as the control group. The pulmonary function [forced expiratory volume in one second (FEV1), forced vital capacity (FVC), FEV1 to FVC (FEV1/FVC) ratio, and percentage of FEV1 to predicted value (FEV1%pred)], neutrophil polarization, and serum total IgE levels of the four groups were compared. Results In the control group, the ACO group, the asthma group, and the COPD group, the FEV1 values were (3.65±0.79), (2.04±0.58), (1.81±0.46), and (1.59±0.43) L, respectively, the FVC values were (4.13±0.92), (3.18±0.76), (2.69±0.63), and (2.43±0.58) L, respectively, the serum total IgE levels were (92.36±12.20), (334.81±55.96), (455.61±65.59), and (142.65±28.36) U/mL, respectively, and the between-group differences were all statistically significant (P<0.05). In addition, the FEV1/FVC ratios in the asthma group, the COPD group, and the ACO group were (67.93±11.51)%, (63.81±9.22)%, and (61.28±9.23)%, respectively, the FEV1%pred levels were (74.55±11.70)%, (63.29±8.60)%, and (61.34±7.91)%, respectively, which were lower than those in the control group [(83.60±7.18)% and (94.23±8.21)%] (P<0.05). The spontaneous polarization rates in the ACO group, the asthma group, the COPD group, and the control group were (29.43±5.58)%, (25.11±4.09)%, (16.28±4.51)%, and (7.18±2.12)%, respectively, the arbitrary polarization rates in the ACO group, the asthma group, the control group, and the COPD group were (30.01±5.29)%, (25.76±5.53)%, (21.42±4.36)%, and (19.85±5.00)%, respectively, the directional polarization rates in the asthma group, the ACO group, the control group, and the COPD group were (14.67±2.30)%, (8.21±1.81)%, (5.12±1.10)%, and (2.52±0.63)%, respectively, and the between-group differences were all statistically significant (P<0.05). Conclusion There are certain differences in lung function, neutrophil polarization, and serum immunoglobulin E level among patients with bronchial asthma, COPD, and asthma-COPD overlap syndrome.
ObjectiveTo investigate the effect of miR-190a-5p on the polarization of bone-marrow-derived macrophage (BMDM) induced by lipopolysaccharides to M1- and M2-types.MethodsBMDM (M1-type) induced by bacterial lipopolysaccharide was a M1 group. The macrophage M1-type interfered with negative control miRNA mimics was a NC group. miR-190a-5p mimics interfered with the M1-type of macrophages in the miR-190a-5p group. Morphological changes of macrophages were observed under a microscope, and the proportion of M2-type macrophages (CD206+, F4/80) was detected by flow cytometry. The mRNA expression levels of argininase-1 (Arg1), inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), target gene C/EBPα and PU.1 were detected by fluorescence quantitative PCR to verify whether C/EBPα and PU.1 were potential target genes of miR-190a-5p. The expression of pathway proteins C/EBPα and PU.1 were detected by Western blotting.ResultsAfter miR-190a-5p mimics interfered with macrophage M1-type, the antenna of macrophages elongated and showed long cord M2-type cell morphological characteristics. miR-190a-5p mimics interfered with M1-type macrophages for 24 h, and the percentage of M2-type macrophages increased significantly (P<0.05). Effects of miR-190a-5p simulator on mRNA expression levels of M1-type macrophages included: the expression of iNOS and TNF-α was significantly decreased (P<0.05), the expression of Arg1 marked by M2 macrophages was significantly increased (P<0.05), and the mRNA expression levels of target genes C/EBPα and PU.1 were significantly decreased (P<0.05). Western blotting results showed that the overexpression of miR-190a-5p significantly inhibited the protein expressions of C/EBPα and PU.1, while the miR-190a-5p inhibitor increased the expressions of both proteins.ConclusionmiR-190a-5p can promote the polarization of BMDM from M1-type to M2-type.
Objective To summary the regulatory effect of mechanical stimulation on macrophage polarization in wound healing, and explore the application prospect of mechanical stimulation in tissue engineering. Methods The related domestic and foreign literature in recent years was extensive reviewed, and the different phenotypes of macrophages and their roles in wound healing, the effect of mechanical stimulation on macrophage polarization and its application in tissue engineering were analyzed. Results Macrophages have functional diversity, with two phenotypes: pro-inflammatory (M1 type) and anti-inflammatory (M2 type), and the cells exhibit different activation phenotypes and play corresponding functions under different stimuli. The mechanical force of different types, sizes, and amplitudes can directly or indirectly guide macrophages to transform into different phenotypes, and then affect tissue repair. This feature can be used in tissue engineering to selectively regulate macrophage polarization. Conclusion Mechanical stimulation plays an vital role in regulating macrophage polarization, but its specific role and mechanism remain ambiguous and need to be further explored.