| Background:The immune system is an important defense system utilized by multi-cellular organisms, especially manmals, against invaders to maintain their health and homeostasis. The immune cells communicate with each other via cytokines, chemokines, and cell-contact to realize their function. The normal immune system maintains a relative balance through positive or negative regulation. Insufficient or excessive immune response can develop diseases. Inflammation result from immune response caused by impair of tissue and infection of pathogen. Appropriate inflammation can clear pathogen and benefit the recovery of host, while diseases can be deteriorated by excessive and chronic inflammation. Sepsis caused by excessive inflammation can develop into system inflammatory response syndrome(SIRS), progressing into multiple organs dysfunction syndrome(MODS) and even death. Chronic inflammation, caused by many factors such as aging,oxidative stress, physical and chemical stimulation, can induce cellular impair and even the dysfunction of organs via activating immune cells continually. In this situation, Host may develop many chronic disease such as hypertension,diabetes, coronary artery disease, hepatic fibrosis, chronic nephritis, tumor,and Alzheimer disease. Therefore, excessive and chronic inflammation is the basis of pathphysiology in most of severe infectious diseases and chronic diseases. It is critical to regulate inflammatory response and restore balance in prevention of these diseases.Cyclophilin A(CyPA) is an ubiquitously distributed protein belonging to the immunophilin family, which plays an important role in a variety of biological processes and is well conserved and present in some prokaryotes and all eukaryotes. There are two relatively independent domains of CyPA separately performing peptidyl- proline cis-trans isomerase(PPIase) activity and nuclease activity. CyPA is believed to have important roles in many biological conditions including protein folding, trafficking, and T-cell activation.Although CyPA is mainly present intracellularly, it can be secreted from cells in response to inflammatory stimuli such ashypoxia, infection, and oxidative stress. The mechanism of CyPA working as a proinflammatory factor unequivocally depended on the combination with CD147 receptor. CD147, a single transmembrane glycoprotein, is widely expressed on the cell surface which can be expressed in all of the white blood cells, platelets and endothelial cells in most normal tissues in weak or no expression. The proline180 and glycine 181 residues in the extracellular domain of CD147 were critical for signaling and chemotactic activities mediated by CD147 which could be accomplished by the PPIase activity of CyPA. The activated CD147 could transfer information into cells, leading to chemotaxis, release of factor and apoptosis of ECs. Since CyPA is widely distributed and play an important role in early inflammation reaction, this protein has been shown to play an important role in many diseases, including tumors, viral infections, diabetes,sepsis, autoimmune diseases, and so on.Currently, the strategies to cure inflammatory diseases are almost all concentracted on mediating inflammatory cells and factors in local inflammatory tissues. As the existence of heterogeneity, there are different pathophysiological processes in different organ systems. So, these strategies almost have some short remission and the disease is easy to recurrent. Is there any common pathophysiology in different tissues and different types of inflammation? The initial factor initiats inflammatory reaction from the source and is the target to effectively prevent and control various types of acute and chronic inflammatory diseases. Based on comprehensive documents, we find a multifunctional protein cyclophilin A(CyPA) might be an inflammatory initial factor.Parasites are unicellular or multicellular eukaryotes which constitute a special symbiotic relationship with the host. On one hand, parasites induce mechanical and chemical damage and cause inflammation response of the host, which develops parasitic diseases; on the other hand, parasites could modulate immune system of the host and reduce immune attack in order to complete their life cycle, which is named immune evasion. Hence, parasitic immunomodulatory effects on the host must be bound to affect the body's inflammatory reaction to other stimuli. In developed countries, the rate of parasitic infection drops to a low level. However, according to the large epidemiological studies, the direct consequence of the elimination of parasites is the increasing morbidity of immune system diseases, diabetes,severe infections and other diseases. There is no improvement on the total health level of people. Thus, the classical understand on the relationship between parasite and host is now questioned. There might be a mutual interest in compromise symbiotic relationship between parasite and host.During infection, parasites enhance the host's ability to mediate immune system. In long-term co-evolution, some parasitic genes convergently evolve with host's genes led to similar structure and function. This phenomenon reduces the immunogenicity of parasitic proteins to achieve immune evasion.Meanwhile, this similarity induces some cross-reactions between parasitic proteins and host proteins. Hense, the antibodies which produce by host to react with parasitic protein could inhibit the pathogenic effects of endogenous homologous proteins. In our previous study, Cs CyPA is a kind of excretory-secretory antigen which could promote the host to produce antibodies. The anti-Cs CyPA antibodies could cross-react with CyPA from host. The aim of this article is to observe whether the anti-Cs CyPA antibodies have a protective effect on acute and chronic inflammatory reactions induced by CyPA.Objective:Through the establishment of sepsis model and pancreatic ? cell damage model to demonstrate the initial role played by CyPA in acute and chronic inflammatory reactions, and to observe whether the anti-Cs CyPA antibodies have a protective effect, in order to understand the effect of proteins from parasites for prevention and treatment of human diseases.Methods:1. Bioinformatics analysis of Cs CyPA, Sj Cyp A, Hs CyPA and Mu CyPAThe amino acid sequences were downloaded from Genbank. The putative PPIase catalytic area was identified by Motif Scan.2. Immunoreactivity between r CyPA and Anti-Cs CyPAsWestern blot analysis demonstrated whether r Cs CyPA, r Sj CyPA,r Mu CyPA and r Hs CyPA could be recognized by anti-Cs CyPAs.3. PPIase Activity and InhibitionColorimetric detection of PPIase activity was performed by the chymotrypsin-coupledcleavage assay according to Fischer et al.4. Prognosis of Sepsis Improved by Anti-Clonorchis Sinensis Cyclopholin A Antibodies4.1Preparation of Polyclonal AntibodiesAntisera were precipitated with ammonium sulphate(33% saturation)and were purified by affinity chromatography on a G-Sepharose column. The concentration of anti-Cs CyPAs was measured by using a BCA Protein Assay Kit.4.2CLP ModelSepsis was induced in the mice model by CLP with a 80% death rate at72 h after surgery.4.3Measured Cytokine and CyPA in SerumMu CyPA were measured using a mouse cyclophilin AELISA Kit. TNF-?,IL-6, IL-1?, IL-4 and IL-10 were determined by the corresponding ELISA Kit.4.4Pathological Observation of Lung and Mesentery TissuesThe paraffin-embedded samples were cut into 5?m thick sections and stained with hematoxylin-eosin.4.5Blood Coagulation IndicatorBlood samples in each subgroup collected by anticoagulant tubes were texted within four hours. Prothrombin time(PT), activated partial thromboplastin time(a PTT) and fibrinogen were detected in an automated coagulometer. Platelet count was performed using an automatic blood cell counter. D-dimer was detected by D2 D ELISA Kit.4.6Vascular Endothelial Cells Isolation, Culture and TreatmentThe isolation of ECs from murine aorta was described by Mika Kobayashi in 2005. Different concentrations of r Mu CyPA and anti-Cs CyPAs were co-cultured with ECs for 72 h before being measured by Cells Counting Kit-8.5. The Protective Effects of Cs CyPA Immunization against the Damage on Mice Islet Beta-cell Induced by STZ5.1Mice ImmunityC57 mice were injected subcutaneously with 50?g r Cs CyPA emulsified with equal volume of complete Freund's adjuvant(CFA, Sigma), followed by three boosts with 25?g antigen emulsified with incomplete Freund's adjuvant(IFA, Sigma) at 2-week intervals.5.2Generation of diabetic modelAfter 4 weeks of high-fat diet feeding, the mice were injected once with STZ toinduce partial insulin deficiency. High-fat diet was feeded for another six weeks.5.3The Change of Plasma Glucose and Body WeightPlasma glucose and body weight was monitored every other week.5.4Oral Glucose Tolerance TestsMice were fasted for 4 h before glucose tolerance tests. Oral glucose load was administered at 2 g/kg of body weight.Glucose levels were measured at 0, 30, 60, 90 and 120 minutes after glucose administration.5.5Immunolabeling of Pancreas Sections and Quantification of Islet Cellmass.The pancreas tissues were fixed in 10% for maldehyde for 48 h and then embedded in paraffin. Sections were immunolabeled with anti-glucagon antibodies and anti-insulin antibodies. Image-Pro Plus6.0 was used to count the cell amount.5.6Measured Cytokines, Insulin and CyPA in SerumThe datas were measured by the corresponding ELISA Kits.Results:1. Bioinformatics AnalysisThe identities of amino acid sequence between Cs CyPA and the other three were 66%, 74% and 74% respectively. The identities of amino acid sequence in catalytic area between Cs CyPA and the other three were ordinal92%, 83% and 83% respectively.2. Immunoreactivity between r CyPA and Anti-Cs CyPAsWestern blot analysis demonstrated that r Cs CyPA, r Sj CyPA, r Mu CyPA and r Hs CyPA could be recognized by anti-Cs CyPAs. The results indicated that r CyPA of these species shared similar immunoreactivity with anti-Cs CyPAs.3. Enzyme Characteristicsand Inhibition Experimentsr Mu CyPA possesses PPIase activity. Antibodies could inhibit enzymatic activity and show significant dose-dependent inhibition.4. Prognosis of Sepsis Improved by Anti-Clonorchis Sinensis Cyclopholin A Antibodies4.1 Survival RatesThe survival rates were analyzed 72 h after the CLP surgery. The survival rate of Sham-surgery group was 100%. In the CLP control 72 h subgroup, the survival rate is 10%. In CLP treatment 72 h subgroup, the survival rate is 45%.A statistically significant difference between the CLP control group and the CLP treatment group was observed(p<0.05).4.2 CyPA Level in SerumMu CyPA in the CLP control group was statistically higher compared with the sham group at 6, 12, 24 and 48h(p<0.05). There was no significant difference at 72h(p>0.05). In the CLP control group, the CyPA level reached a climax at 6h followed by a time-dependent decrease.4.3 Cytokines Levels in SerumAdministration of antibodies significantly reduced the levels of TNF-?,IL-6 and IL-1? in serum compared with CLP control mice at 12 h and 24 h after CLP. Adjustment Cytokine had no statistical meaning.4.4 Histology of Lung and Mesentery Tissues4.4.1 Lung TissueLung tissue specimens in the CLP control group presented apparent thrombus at 12 h, and were characterized by leukocyte influx, edema,hemorrhage, wall thickening and alveolar consolidation at 48 h and 72 h. In contrast, lung tissue in the CLP treatment group presented apparent thrombus at 24 h, and demonstrated leukocyte influx and edema without obvious hemorrhage and alveolar consolidation at 48 h and 72 h.4.4.2 Mesentery TissueThe mesentery tissue in the CLP control group demonstrated more serious inflammatory reaction. Tons of inflammatory emigrated from vessel at6h. The boosting of cytolysis, fusiform cells and intercellular substance led to diffuse mesentery fibrosis at 72 h after surgery. In the CLP treatment group,inflammation was limited and some normal fat cells could still be observed at72 h.4.5 Effects of Antibodies on Blood Coagulation IndicatorThe CLP model successfully induced DIC. Compared with the sham group, the platelet count, fibrinogen concentration, PT, a PTT and D-dimer in the CLP control group began to change significantly at 6h post-CLP.Comparing the CLP control group with the CLP treatment group, there were significant differences on the indicators including PT, a PTT, D-dimer and platelet count. Furthermore, antibodies in mice obviously ameliorated these four indicators at 48 h and at 72 h after the surgery.4.6 Viability of ECs with Mu CyPA and Anti-Cs CyPAs by CCK-8 assaysCompared with the control group, the viability of ECs exposed to r Mu CyPA showed a dose-dependent decrease. Compared with 10?g Mu CyPA group, the result showed significant increase on the percentage of cells in the 1?g and 10?g antibody groups in a dose-dependent manner(p<0.05). There was no significant difference between 10?g Mu CyPA group and 0.1?g antibodies group(p>0.05).5. The Protective Effects of Cs CyPAImmunization against the Damage on Mice Islet Beta-cell Induced by STZ5.1 The Change of Body WeightThe mean body weights of mice in PBS Immunity and CyPA Immunity groups kept getting down since STZ injecton and gradually lower than the normal group. Compared with PBS Immunity group, the mice in CyPA kept a higher weight.5.2 The Change of Plasma GlucoseThe plasma glucose levels of mice in normal group remained at 9. After STZ injection, the plasma glucose levels of mice in PBS Immunization group and r Cs CyPA immunization group were significantly reduced. Compared with PBS Immunization group, mice in r Cs CyPA immunizationgroup remained lower level of glucose before injection and then slowly decreased after injection.5.3 Oral Glucose Tolerance TestsThe plasma glucose levels of mice in three groups significantly increased with the similar amplitude, and then began to decline. The plasma glucose levels of mice in normal group returned to normal at 120 minutes after the test.The plasma glucose levels of mice in r Cs CyPA immunization group and PBS Immunizationgroup all decreased slowly. At 120 minutes, the plasma glucose levels of mice in r Cs CyPA immunization group was still elevated 14%compared to fasting blood-glucose. However, the rate in PBS immunization group is 100%.5.4 Immunolabeling of Pancreas Sections and Quantification of Islet Cellmass.As expected, islets of normal diet–fed control mice comprised a large insulin positive ?-cell core surrounded by a mantle of glucagon positive?-cells.In contrast, islets from mice in PBS immunization group contained many more glucagon positive ?-cells, which infiltrated the entire islet including the central core.There was no significant difference in the amount of ?-cells between normal group and 2w r Cs CyPA immunization group. There were significant differences in the amount of ?-cells between PBS immunization group and r Cs CyPA immunization group in 4w and 6w.5.5 CyPA in SerumAfter STZ injection, compared with normal group, CyPA level in PBS immunization group significant increased in all time parts. There was no significant difference of CyPA levels between r Cs CyPA immunization group and normal group.5.6 Insulin in SerumCompared with normal group, mice in PBS immunization group at 2w, 4w and 6w and in r Cs CyPA immunization at 4w and 6w all significant decreased insulin levels in serum. There were significant differences of insulin levels in every time part between r Cs CyPA immunization group and PBS immunizaiton group.5.7 Cytokines in SerumThere was similar trend of IL-1? and TNF-? in r Cs CyPA immunization group and PBS immunization group, which implied chronic inflammation state in there two group. Overall, in r Cs CyPA immunization group, early chronic inflammation could be inhibited and. However, there was no significant difference at 6w between PBS immunization group and r Cs CyPA immunization group.Conclusions:1. Anti-CsCyPAs could recognize and cross-react with Mu CyPA and effectively inhibit the PPIase enzymic activity as the obvious identity of CyPA amino acid sequence in different species.2. Anti-Cs CyPA antibodies had a verified beneficial effect on sepsis induced by CLP surgery. The indicators of pathology, cytokines, blood coagulation indicator and survival rate are all generally improved.3. Anti-Cs CyPA antibodies had a verified beneficial effect on ?-cells damage induced by STZ injection. The indicators of islet ?-cells amount and insulin secretion all generally improved. The chronic persistent inflammation statement was significant improved in CyPA Immunization Group. |
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