The protective role of interleukin-6 monoclonal antibody on experimental autoimmune myocarditis and its mechanism / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the therapeutic effect of IL-6 mAb on experimental autoimmune myocarditis (EAM) in rats, and search the mechanism of the role of IL-6, helper T cells 17 (Th17) and regulative T cells (Treg) in EAM pathogenesis.</p><p><b>METHODS</b>Thirty-four Lewis rats were divided into three groups randomly, i.e. control group (n = 6), EAM group (n = 12), and IL-6 mAb intervention group (n = 16). Rats in EAM group and IL-6 mAb intervention group were injected intracutaneously with myosin to establish EAM model. Rats in IL-6 mAb intervention group were injected intraperitoneally with 1 mg IL-6 mAb on 1st, 7th to 20th day after cardiac myosin immune injection. Myocardial inflammation was examined by HE stain, Masson stain, and TdT assay (TUNEL reaction) on 21st and 84th day after IL-6 mAb therapy in order to assess the therapeutic role. Spleen cells were analyzed by flow cytometry to illustrate Th17 and Treg cells? number and function. The serum concentration of IL-6, IL-10, IL-17, and TGF-beta in each group was measured by ELISA, concentration of STAT3, RORgammat, and Foxp3 mRNA in each group was determined with RT-PCR. Spleen cells derived from EAM were stimulated by IL-6 mAb in vitro, and the concentration of IL-10, IL-17 and TGF-beta was measured by ELISA.</p><p><b>RESULTS</b>Inflammation score, fibrosis score, and apoptosis index in IL-6 mAb intervention group were significantly decreased as compared with those in EAM group (P < 0.01). The number of Th17 and Treg cells in EAM group on the 21st day (experimental acute peak stage) were increased, and those in intervention group on the 21st day were significantly inhibited (P < 0.01). The concentration of serum IL-6, IL-10, IL-17 and TGF-beta in intervention group on the 21st day was decreased dramatically in comparison with that in EAM group on the same day (P < 0.01). The levels of peripheral blood STAT3, RORgammat, Foxp3 mRNA in intervention group on the 21st day was decreased significantly as compared with that in EAM group (P < 0.01). The expression of IL-10, IL-17 and TGF-beta was increased significantly (P < 0.01) by stimulation of IL-6 mAb on spleen cells derived from EAM in vitro.</p><p><b>CONCLUSIONS</b>IL-6 mAb could neutralize IL-6, and ameliorate myocarditis and reduce heart autoimmune responses. IL-6 mAb has significantly protective effects on EAM by suppressing Th17 and Treg cells.</p>

Analysis of changes in percentage of phenotype CD4 + CD45RA + and CD4+ CD45RO + in peripheral blood and effect of immunomodulation in aged male patients with chronic cardiac insufficiency / 中国应用生理学杂志

<p><b>OBJECTIVE</b>Autoimmunity participates in chronic heart failure (CCI), it is CD4+ T lymphocytes that mainly induces myocardial infiltration and the progression of the disease. The purpose of this research is to assess changes of CD4+, CD8+ T lymphocyte subset, and phenotype of primary T cell (CD4+ CD45RA+) and memory T cells (CD4+ CD45RO+) in peripheral blood in aged male patients with CCI. And to investigate the immunomodulatory effects on subsets of CD4+, CD8+ and phenotype of CD4+ CD45RA+ and CD4+ CD45RO+ and the possible therapeutic mechanism.</p><p><b>METHODS</b>The participant were 155 aged men among whom 94 cases were diagnosed as CCI and heart function of the rest 41 cases were normal. All patients underwent echocardiography examination and were collected peripheral blood before and after treatment. Serum N terminal pro-brain natriuretic peptide (NT-proBNP) levels were detected by heterogeneous immunoassay. Serum C reactive protein (CRP) were measured by immunoturbidimetry assay. T lymphocytes in peripheral blood were separated and determined distribution of CD4+, CD8+, CD4+ CD45RA+, CD4+ CD45RO+ using flow cytometry. Participants were divided into 3 groups: the CCI intervention group, who received regular therapy and thymopentin (20 mg intramuscular injection, once every other day for 3 month; n = 60) , the CCI control group, who received regular therapy (n = 54) and 41 healthy individual older than 57 years of age, who served as normal controls.</p><p><b>RESULTS</b>Compared with the control group, left ventricular ejection fraction (LVEF) and CD4+ CD45RO+ levels decreased, left ventricular end diastolic diameter (LVEDD), NT-proBNP, CRP, CD4+, CD4+ CD45RA+, CD4+/CD8+, CD4+ CD45RA+/CD4+ CD45RO+ levels were obviously higher in CCI group. Distribution of CD8+ was not significantly changed. The level of NT-proBNP, CRP, CD4+/CD8+, CD4+ CD45RA+/CD4+ CD45 RO+ was negatively correlated with LVEF. LVEF could be much improved via decreasing distribution of CD4+/CD8+, CD4+ CD45RA+/CD4+ CD45RO in CCI intervention group than in CCI control group.</p><p><b>CONCLUSION</b>The changes of CD4+/CD8+ and CD4+ CD45RA+/CD4+ CD45RO+ suggest that CD4+ T lymphocyte subset and its phenotype play an important role in the process of CCI. The regulation of CD4+ T lymphocyte and its phenotype may be one of the strategy in the treatment of CCI.</p>

Study of L-NAME treatment on experimental autoimmune myocarditis / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the therapeutic effect of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, on experimental autoimmune myocarditis (EAM) in Balb/C mice and discuss the therapeutic mechanism induced by apoptosis.</p><p><b>METHODS</b>Thirty male Balb/C mice were divided into normal control group, model control group and experimental group randomly (n = 10). Model control group and experimental group were created into EAM by injection of porcine cardiac myosin subcutaneously in double groin and axilla and pertussis toxin intraperitoneally on day 0 and 7 respectively. Model control group was intraperitoneally administered 5 mg/(kg x day) of physiological saline after infective myosin and pertussis toxin. Experimental group was intraperitoneally given 5 mg/(kg x day) of L-NAME on day 1-21. The hearts and blood were processed after sacrificed on day 21. Cardiac inflammation score was measured by HE staining. Heart weight / body weight (HW/BW), serum nitric oxide (NO) level, activity of induced nitric oxide synthase (iNOS) and mRNA expression of iNOS in heart were measured in each group. Degree of heart apoptosis were evaluated by cardiac apoptotic index through TUNEL, immunohistochemical examination and real time PCR of Caspase-3, Caspase-8 and Caspase-9.</p><p><b>RESULTS</b>Compared with normal control group, cardiac inflammation score, HW/BW level of NO and activity of iNOS, mRNA expression of iNOS, the levels of mRNA and protein of Caspase-3, Caspase-8 and Caspase-9 and cardiac apoptotic index were significantly higher (P < 0.01) in model control group, and those of model control group were higher than those of experimental group (P < 0.01). HW/BW was only a little elevation in model control group compared with that in the experiment group (P < 0.05).</p><p><b>CONCLUSION</b>The development of EAM is related with the NO catalyzed by iNOS. L-NAME protects cardiac myocyte via suppressing the activity of iNOS and further decreased production of NO in EAM. The mechanism might be that L-NAME alleviated myocardial inflammation through inhibited the apoptosis of cardiac myocyte.</p>

Establishment of human cardiac C protein induced experimental autoimmune myocarditis model in rat / 中华心血管病杂志

<p><b>OBJECTIVE</b>To construct the recombinant plasmid of human cardiac C protein (CCP) peptide with immunogenicity and to express, purification and renature fusion protein. The fusion protein was injected to Lewis rats to establish experimental autoimmune myocarditis (EAM) model.</p><p><b>METHODS</b>Total RNA was extracted from human heart and used as the template for reverse transcriptase-directed cDNA synthesis. The cDNA was then amplified by polymerase chain reaction (PCR) using oligonucleotide primers specific for CCP peptide with immunogenicity. Subsequently, the purified CCP peptide gene was cloned into PEASY-T1 vector and the ligated product was identified by PCR and DNA sequence analysis. Then the CCP target gene of positive clone was inserted into the pQE30, a prokaryotic expression vector, and the inserting plasmid was transformed into Escherichia coli. host M15. The positive clone extracted from the bacterium liquid was sieved by insertional inactivation sieve method and identified by PCR of bacterium liquid, CCP immunological peptide was purified and renatured in semipermeable membrane. EAM model in Lewis rats was induced by injection of mixture of 100 µg CCP fusion protein immunological peptide and 2.5 g/L completed Freund adjuvant from two double foot pad and subsequent abdominal injection of 0.5 µg pertussis toxin. Two, four, six, and eight weeks after immunization, hemodynamic evaluation was made and hearts underwent histological examination.</p><p><b>RESULTS</b>The DNA sequence analysis for cloning vector extraction revealed that the CCP target gene was cloned into pQE30 exactly. The DNA of 1000 bp length was obtained by PCR examination of bacterium liquid with transformation of express recombinants which were consistent with the expected size. Purified fusion protein in vertical slab gel electrophoresis showed 35 000 as expected. The recombinant CCP fusion protein existed in inclusion bodies of E. coli and amounted to 80% - 90% of the total protein. Hemodynamic and histological evaluations showed typical acute inflammatory responses at 2 weeks, subacute inflammatory and fibrosis changes at 4 weeks after injection, and signs of chronic dilated cardiomyopathy at 6 weeks post injection.</p><p><b>CONCLUSION</b>Combination of gene clone technique and histidine tag protein purification technique can be used to synthesize human cardiac C protein to induce EAM model in Lewis rat.</p>