Studies On The Role Of Calpain-2in The Doxorubicin-induced Cardiotoxicity And The Development Of RagB-mGITRL DNA Vaccine

Myocardial injury was mediated by toxicity of chemical and myocarditis caused by infection of virus, bacteria and so forth. At same time, it seriously affected our health. In order to avoid disadvantages and prevent heart from pathological damage, this thesis involved the intervention study for two types of cardiac toxicity induced by doxorubicin and Porphyromonas gingivalis.Doxorubicin, as an anthracycline antibiotic intercalating into DNA, is widely used in the clinical treatment of tumor. Doxorubicin may bring some side effects such as nausea, vomiting and arrhythmia. Sometimes, it may cause congestive heart failure, dilated cardiomyopathy and even death when its cumulative dose reaches55mg/m2, for which its clinical application is limited. However, the mechanism of acute and cumulative doxorubicin cardiotoxicity has not yet been fully understood, nor is there any effective prevention or treatment of doxorubicin-induced cardiomyopathy.Calpain which is widely expressed in mammalian and other biological varieties belongs to the family of calcium-dependent cysteine protease. There are already15subtypes of calpain respectively encoded by15genes have been identified by human, with distinct differences in distribution, composition and function of each subtype. In which the u-calpain (calpain-1) and m-calpain (calpain-2) are very common with intensive studies. The activation of calpain requires Ca2+. Its endogenous inhibitor is calpastatin which is the specific inhibitor of calpain. The activation of calpain is involved in many systematic diseases of the body, such as embryonic lethal, diabetes, Alzheimer’s disease, myocardial infarction, heart ischemia-reperfusion injury, atherosclerosis and gastric cancer, etc. Our previous study showed that over-expression of calpastatin increased acute myocardial toxicity induced by doxorubicin, indicating calpain was closely related with acute myocardial toxicity induced by Adriamycin. Was calpain-1or calpain-2or both involved in the protective effect and in which way? How was the molecular mechanism? These are questions still need to be answered.Atherosclerosis, another important disease of cardiovascular system, has multiple incentives in which paradentitis is a major one. Porphyromonas gingivalis is the main pathogenic bacteria of paradentitis and it also participates in and promotes the formation of foam cells. Considering this, will prophyromonas gingivalis vaccine which can effectively prevent the paradentitis also prevent atheroscelrosis? And with adjuvant GITRL, will it be more efficient in cleaning up the invading pathogenic microbes? Will it be a catalyst in anti-infectious immunity? There are more to be proved.Purposes(1) Our previous studies found out that in the model of injecting doxorubicin into the primary myocardial cells and abdominal cavity of mouse, causing acute myocardial toxicity, the expression and activity of calpain was lowered in its heart. In transgenic mouse with over expression of calpastatin, the injection of doxorubicin into abdominal cavity inducing acute myocardial toxicity exacerbated the mouse’s cardiac dysfunction and increased mortality because the activity of calpain is inhibited. The above studies indicated that endogenous calpain provided a protection against myocardial toxicity induced by doxorubicin. However, the subtype of calpain that was involved in this protection is not so clear. Herein, our study was to explore the effect of calpain-2in the myocardial toxicity induced by doxorubicin and its possible molecular mechanism.(2) In order to further study the effect of calpain-2in the myocardial toxicity induced by doxorubicin, a cardiac specific and inducible over-expression transgenic system of calpain was constructed. In transgenic mouse, we used doxorubicin to induce acute and chronic myocardial toxicity models and then studied the calpain-2’s effect and mechanism in both models.(3) Studies have shown that the occurrence and development of atherosclerosis are closely related with paradentitis of which the Porphyromonas gingivalis is the major pathogenic bacteria and involves in the occurrence of atherosclerosis. At the same time, paradentitis is also the incentive of multiple diseases like aspiration pneumonia, chronic kidney disease and diabetes, etc. Therefore, our study was to enhance the protection against Porphyromonas gingivalis’infection and reduce the incidence of paradentitis through preparing a DNA vaccine of outer membrane protein RagB of Porphyromonas gingivalis with adjuvant GITRL, thus to control the occurrence and development of multiple diseases such as atherosclerosis. Also, it was to further study the DNA vaccine’s protective effect and molecular mechanism against the infection of Porphyromonas gingivalis to provide theoretic basis for DNA vaccine’s future clinic application.Methods(1) Establishment of cardiac-specific and inducible calpain-2transgenic miceHuman calpain-2(hcalpain-2) nucleic acid sequence contained TetO and a-MHC promoter sequence at5’end and hGH sequence at3’end was obtained with PCR, and cloned into the eukaryotic expression vector. Recombinant plasmid was imported into mouse fertilized egg with microinjection, which implanted in womb of mice, and developed into hcalpain-2transgenic mice. Selecting hcalpain-2-positive mice were mated with tTA transgenic mice, and the offspring was genotyped with PCR. Hcalpain-2activity in the myocardium was detected with zymography. Western blot method was employed to assess hcalpain-2and calpain-1protein level in the heart or lung of transgenic mice. The heart function of mice was measured with Doppler method.(2) Establishment and analysis of mice model with doxorubicin-induced cardiotoxicityTransgenic mice identified with genotyping were divided into two groups, that is, control group and doxorubicin-treated group. At least four mice in each group and6-8mice in Tg-tTA/capn-2group with doxorubicin treatment. Doxorubicin-induced acute cardiotoxicity model:12-week old male mice were given doxorubicin by intraperitoneal injection at each genotype, and control group mice were given sterile PBS by intraperitoneal injection. At the same time, taking another six Tg-capn-2and Tg-tTA/capn-2mice respectively were treated with calpain inhibitor (CⅠ-Ⅲ) by intraperitoneal injection. Doxorubicin-induced chronic cardiotoxicity model:8-week old male mice were given doxorubicin by intraperitoneal injection according to the weight of mice; control group mice were given sterile PBS. Heart function of mice was measured with Doppler and hearts were harvested for subsequent experiments.(3) To assess the protection mechanisms of calpain-2in doxorubicin-induced cardiotoxicitySeparating neonatal mouse cardiomyocytes were treated, as follows: Cardiomyocytes were infected with adenovirus calpain-2and HA respectively and given lμM doxorubicin. Caspase-3activity was detected with AMC substrate method in cardiomyocytes. ELISA method was employed to detect DNA fragmentation level and phosphorylated AKT was measured with western blot. Cardiomyocytes were infected with adenovirus calpastatin and HA respectively and given lμM doxorubicin. Caspase-3activity of cardiomyocytes was detected with AMC substrate method. ELISA method was employed to detect DNA fragmentation level and phosphorylated AKT was measured with western blot.Myocardial tissue of mice in acute cardiotoxicity model was used for measuring caspase-3activity with AMC substrate method and detecting the phosphorylated AKT by western blot. The hearts of mice were embeded with wax, stained with WGA and measured the cell size with imageJ software in doxorubicin-induced chronic myocardial toxicity model.(4) Construction and expression in vitro of Porphyromonas gingivalis DNA vaccineRagB and mGITRL nucleic acid sequences were acquired with PCR from plasmid contained RagB and mGITRL respectively and cloned into the vector pIRES. Getting recombinant, that is, pIRES-ragB and pIRES-ragB-mGITRL. The constructed recombinant was transfected into COS-7cells, and then western blot was used to detect ragB and mGITRL expression.(5) The role and molecular mechanism of the outer membrane protein ragB DNA vaccine and adjuvant mGITRL against Porphyromonas gingivalis infectionSix-week female Balb/c mice were immunized with recombinant by intramuscular injection in the three groups, that is, pIRES-ragB, pIRES-ragB-mGITRL, and pIRES. Mice immunized for six weeks, were infected with1×109Porphyromonas gingivalis by subcutaneous injection to induce ulcer model. After48hours, measuring the ulcer area. The titer of anti-RagB antibody in serum was detected with ELISA. ELISPOT assay was employed to measure the number of specific antigen-forming cells in the spleen cells and bone marrow cells. IL-21and IFN-γ mRNA level in spleen were assessed by QRT-PCR and Tfh cells and IFN-γ+T cells ratio in spleen were detected with FCM.Results(1) Acquiring hcalpain-2fragment with TetO and a-MHC promoter at5’end and hGH nucleic acid sequence at3’ends, and have been cloned into eukaryotic expression vector. The constructed plasmid was introduced into mouse fertilized eggs, which was implanted in the uterus of the recipient mice, and developed into hcalpain-2transgenic mice. At last, we acquired four genotypes of mice, that is, WT, Tg-capn-2, Tg-tTA and Tg-tTA/capn-2. Western blot analysis showed hcalpain-2was inducible and cardiac-specific expression and had no effect on endogenous calpain-1and calpain-2protein levels in other organs. Zymography results indicated that hcalpain-2over-expression in the myocardium increased the activity of calpain-2. Detection of cardiac function showed no difference in systolic and diastolic function among four genotypes of mice.(2) Establishment of doxorubicin-induced acute and chronic cardiotoxicity model, and measurement result of cardiac function showed that:In the acute myocardial toxicity model, doxorubicin-treated group (WT, Tg-capn-2and Tg-tTA groups) mice were significantly reduced the systolic function of heart compared with sham group. However, owing to calpain-2over-expression, systolic function of mice was improved significantly. But given the calpain inhibitor (CⅠ-Ⅲ), the protection of calpain-2was canceled immediately. In the chronic myocardial toxicity model, doxorubicin treatment group (WT, Tg-capn-2and Tg-tTA groups) also were obviously reduced systolic and diastolic function of mice compared with sham group, but the heart function of mice was improved significantly attributing to calpain-2over-expression.(3) At the calpain-2over-expression of myocardial cells treated with doxorubicin, caspase-3activity was significantly decreased comparing with control group; DNA fragmentation was also significantly improved; the level of phosphorylated AKT was significantly up-regulated. However, calpain-1over-expression in primary cardiomyocytes increased caspase-3activity and DNA fragmentation. At the effect of calpain physiological inhibitor (CⅠ-Ⅲ and PD150606) and endogenous inhibitor (calpastatin), caspase-3activity and DNA fragmentation of cardiomyocytes treated with doxorubicin were significantly increased comparing with sham group. Calpastatin over-expression in myocardial cells downregulated the AKT phosphorylation comparing with control group after treating with doxorubicin. In the heart tissue of doxorubicin-induced acute and chronic cardiotoxicity model, the caspase-3activity was increased comparing with sham group and canceled owing to calpain-2over-expression. At the same time, over-expression of calpain-2increased AKT mRNA and phosphorylated protein levels. Also, calpain-2over-expression significantly improved doxorubicin-induced myocardial hypertrophy.(4) PCR amplified ragB, and mGITRL fragment, and cloned into the eukaryotic expression vector pIRES. Acquired recombinants pIRES-ragB and pIRES-ragB-mGITRL. Recombinant pIRES-ragB-mGITRL was transfected into COS-7cells, and western blot results displayed two specific bands, RagB and mGITRL.(5) Results of ELISA assay showed that mice immunized with ragB DNA vaccine produced high titers of RagB specific antibody (1:32000), while given adjuvant GITRL, produced higher titer specific antibody (1:64000). ELISPOT analysis results showed that RagB specific antigen-forming cells were produced in the spleen cells and bone marrow cells of mice immunized with ragB DNA vaccine, with the effect of adjuvant GITRL, RagB specific antigen-forming cells were increased by more than three times. IL-21and of IFN-y mRNA in the spleen and protein in serum of mice immunized with ragB DNA vaccine were significantly raised, while given GITRL further increasing the levels. The proportion of Tfh and IFN-γ+T-cells in CD3+cells of spleen significantly up-regulated due to the application of ragB DNA vaccine, and the adjuvant GITRL further raised the amplification of two cells. The application of DNA vaccine significantly decreased lesion area caused by Porphyromonas gingivalis via abdominal infection, and the participation of adjuvant, further narrowing the lesion size.Conclusions(1) Eukaryotic expression vector contained hcalpain-2sequence with TetO, a-MHC promoter and hGH nucleic acid sequence was successfully constructed and imported into mouse fertilized eggs, which produced a transgenic mice carried hcalpain-2gene. And then mating with tTA transgenic mice produced an offspring with calpain-2cardiac-specific and inducible expression. Hcalpain-2over-expression in heart unaffected function of various organs in transgenic mice.(2) Doxorubicin-induced mice acute and chronic cardiotoxicity model were successfully constructed. Calpain-2over-expression providing protection against doxorobicin induced cardiac toxicity is expected to become a target for the prevention and treatment of doxorubicin induced cardiotoxicity, thereby enhancing doxorubicin anticancer applications.(3) Calpain-2improved AKT mRNA and protein phosphorylation levels in primary myocardial cells in vitro and myocardial tissue in vivo. Calpain-2provided protection against doxorubicin induced cardiac toxicity via AKT pathway.(4) The successful construction of eukaryotic expression vector, pIRES-ragB mGITRL and pIRES-ragB-mGITRL, which successfully expressed in COS-7cell line. (5) Mice immunized with DNA vaccine produced anti-RagB antibody and provided a protective effect for Porphyromonas gingivalis infection, while the application of adjuvant GITRL further improved the protection efficiency of the vaccine. The adjuvant GITRL increased the quantity of the RagB specific antibody-forming cells in the spleen and bone marrow by raising the proportion of Tfh cells in spleen, which should further improved the production of specific antibodies. Meanwhile, the GITRL increased the ratio of IFN-γ+T cells in spleen. In summary, GITRL played a important role in enhancing protection function of ragB DNA vaccine against Porphyromonas gingivalis infection via two ways: The Tfh cells and the IFN-γ+T cells. Outer membrane protein ragB DNA vaccine was hopefully to prevent Porphyromonas gingivalis infection as well as the occurrence of periodontitis, thereby reducing the occurrence and development of atherosclerosis, diabetes and other diseases. The GITRL was expected to be the major candidate for vaccine adjuvants.