| LPS of dominant bacteria of periodontitis is a potential cell activator which can activate many kinds of target cells in periodontal tissues, such as immunologic reaction cells (macrophage and Lymphocyte), neutrophils which having defensive function, major cells of periodontal tissues (fibroblast), osteoblasts and osteoclast, etc. LPS can also make these cells synthesize and secret various kinds of cytokines and inflammatory mediators for the damage of periodontal tissues.Study showed that LPS can activate monocyte-macrophage to produce cytokines and inflammatory mediators with low concentration of Ipg/ml, but a single factor of microorganism will not develop periodontal diseases. The recent study showed a susceptible host is the essential element of occurrence of periodontal diseases. Some researchers even take this as the decisive factor. Hosts may take different responses to the challenge of bacteria which will be regulated by whole-body factors and influenced by environmental factors. High-grade inflammatory reactions in some hosts will occur and massive cytokines will be released because of some bacteria and LPS. Some whole-body factors such as endocrine disorder, immune deficiency, pressure, heredity and malnutrition may affect the host defense system and decrease the defense ability and repair ability, or they can aggravate the inflammatory reaction and damage of periodontal tissues.Stress is an integrated responsive state including responses of mind, nerves, endocrine and immunity. This is proposed by Han Salye in 1930's. Stress occurs when the body is excited by intensive changes of external environment and it is a result of interaction of internal and external environments. The modern stress theory considered that stress belongs to response of defense adaptation, and it is obtained in order to defend damage through interaction between body and environment factors during the long evolution process. Proper stress can promote the internal protective mechanism and non-specific adaptation system of the body and increase defense of the body to interference factors in internal and external environments and immune ability. For excessive stress, for example, the stressor is too strong and persistent, although the responses of the body can still make certain defense adaptation, the more outstanding responses including tissue damage and disorder of body function and metabolism will be disadvantaged to the body. With further study, stress is defined as absonant state of the body or threat of the stability of internal environment. The main feature of stress is that the activity of sympathetico-adrenomedullary and hypophysis-adrenal cortex system will be enhanced after excitation of the internal and external environments. Chronic stress is defined as continuous or repeated stress which is more than 24h. The state is because of persistent excitation and it is mainly attended by changes of nerves, endocrine, immunity and behavior.According to the previous study, we found there are many studies about secretion of immunologic mediators to endotoxin of porphyromonas gingivalis under stress in previous literatures, but study of the molecular mechanism is only stay at the level of theory derivation. It is necessary to make further discuss to this imagination. Considering of this, the conduction mechanism of Pg-LPS to macrophages in restraint stress rats is investigated in this study. The study includes 4 sections as follows:Part 1 Study on the regulation function of Pg-LPS and restraint stress to gene expression of macrophages in rats with gene chip technology[ObjectiveJTo study the regulation function of Pg-LPS and restraint stress to gene expression of macrophages in rats with gene chip technology and analyze the conditions of cell pathway activation. [Methods] 12 male SD rats with SPF grade were used in this study. They were randomized into 4 groups, i.e. group A (normal control group), group B (stress group), group C (Pg-LPS group) and group D (stress+ Pg-LPS group). Abdominal macrophages were extracted respectively, then RNAs were drawn and cDNAs were got after reverse transcription. The gene chip (MM008, Superarray) for detection of signal pathway were used to detect the 18 signal pathways. [Results] The activation conditions on different pathways in different groups of macrophages: (l)Stress pathway : Up-regulation of genes c-fos, Hsfl, Hsp25, Hsp90 and c-JUN in group C is more than twice of that in group A. Up-regulation of genes c-fos, Hsfl, Hsp25, Hsp90, c-myc and c-Jun in group B is more than twice of that in group A. Up-regulation of genes c-fos, Hsfl, Hsp25, Hsp90, c-Jun and c-myc in group D is more than twice of that in group A. (2)JAK-STAT pathway: Up-regulation of genes Beta-casein, 11-4,11-4R, IRF-1 and Stromelysin2 in group B is more than twice of that in group A. Up-regulation of genes Beta-casein, 11-4, I1-4R, IRF-1 and Stromelysin2 in group C is more than twice of that in group A. Up-regulation of genes 11-4,11-4R, IRF-1, Stromelysin2 and iNos in group D is more than twice of that in group A. (3) Calcium and PKC pathway: Up-regulation of genes GM-csf, c-fos, CD25, c-JUN, c-myc, PKC alpha in group B is more than twice of that in group A. Up-regulation of genes c-fos, CD25, c-JUN, PKC alpha in group C is more than twice of that in group A. Up-regulation of genes GM-csf, c-fos, CD25, c-JUN, c-myc, Ornithine decarboxylase in group D is more than twice of that in group A. (4) Phospholipase C(PLC) pathway: Up-regulation of genes Krox-24, c-fos, ICAM-1 and c-JUN in group B is more than twice of that in group A. Up-regulation of genes Krox-24, c-fos and c-JUN in group C is more than twice of that in group A. Up-regulation of genes Krox-24,c-fos, c-JUN, iNOS and COX-2 in group D is more than twice of that in group A. (5) P53 pathway: Up-regulation of genes bax, Igftbp3, Fas and FasL in group B is more than twice of that in group A. Down-regulation of Gadd45 is more than twice of that in group A. Up-regulation of genes bax and PIG8 in group C is more than twice of that in group A. Up-regulation of genes bax, Igftbp3, Fas, Trail, FasL and p53 in group D is more than twice of that in group A. (6)NF-kB pathway: Up-regulation of genes ICAM-1, TNFb and NAIP2 in group B is more than twice of that in group A. Up-regulation of genes TNFb in group C is more than twice of that in group A. Up-regulation of genes TNFb, NAIP2, NAIP1, TNFa, ikBa and iNOS in group D is more than twice of that in group A (7) Androgen pathway and Retinoic acid pathway: Up-regulation of genes Hoxal, Hoxbl, Stra6 in group B is more than twice of that in group A. Down-regulation of kallikrein6 is more than twice of that in group A.Up-regulation of genes Hoxbl,EGFR in group C is more than twice of that in group A. Up-regulation of genes Hoxal,Hoxbl, CRBPII, Stra8, EGFR and kallikrein3 in group D is more than twice of that in group A. Down-regulation of Cdxl is more than twice of that in group A [Conclusion] Pg-LPS and stress can activate many genes on pathways of stress, such as Jak-Stat, Calcium and PKC and PLC. At the same time, they can still activate many genes on pathways of P53, NF-kB, androgen and retinoic acid. When the two factors are combined, the activation of pathways is more obvious, especially for the expression of genes related to cell apoptosis.Part 2 Regulation function of Pg-LPS to expression of genes related to cell apoptosis of macrophages in restraint stress rats with real-time PCR[Objective] Study on the regulation function of Pg-LPS to expression of genes related to cell apoptosis of macrophages in restraint stress rats with real-time PCR. [Methods] 12 male SD rats with SPF grade were used in this study. They were randomized into 4 groups, i.e. group A (normal control group), group B (stress group), group C (Pg-LPS group) and group D (stress+ Pg-LPS group). Abdominal macrophages were extracted respectively, then RNAs were drawn and cDNAs were got after reverse transcription. Real-time PCR was used to quantitative determined the mRNA level of genes Bax, FasL, TNF-a and Trail. [Results] (l)Bax mRNA: Bax mRNAs in group B and C are significantly higher than group A (P<0.01) . Bax mRNA in group D is not only significantly higher than group A (P<0.01) , but also higher than group B and C (P<0.01). (2) Trail mRNA: Trail mRNAs in group B and C are significantly higher than group A (P<0.01), Trail mRNA in group D is significantly higher than other 3 groups (P<0.01) . (3) TNF-amRNA: TNF-a mRNAs in group B and C have no significant difference compared with group A, but TNF-amRNA in group D is significant higher than group A and C (p<0.01). (4) FasL mRNA: FasL mRNAs in group B and D are significantly higher than group A and C ( P <0.01) .[Conclusion] Pg-LPS can increase the expression of Bax and Trail mRNAs. Stress can increase the expression of Bax, Trail and FasL mRNAs. When the two factors are combined, expression of Bax, Trail, FasL and TNF-a mRNAs can all increase. This showed that combination of the two factors might promote apoptosis.Part 3 Effect of Pg-LPS on protein Bax and caspase-3 in macrophagesin stress rats[ObjectiveJFurther verification of the cell apoptosis in each group on the basis of gene chip and real-time PCR. [Methods] 20 male SD rats with SPF grade were used in this study. They were randomized into 4 groups, i.e. group A (normal control group), group B (stress group), group C (Pg-LPS group) and group D (stress+ Pg-LPS group). Abdominal macrophages were extracted respectively, then Elisa was used to detect the activity of Caspase-3 and Western blot was used to detect the expression of protein Bax. [Results] (1) Results of Caspase3 showed: Caspase3 increased significantly in group C compared with group A (p < 0.01) and it also increased significantly in group B compared with group A (p < 0.01) . It increased significantly in group D compared with other 3 groups (p<0.01);(2) Western Blot showed: There is no difference between group C and A (p>0.05), Bax in group B increased significantly compared with group A (p<0.05), Bax in group D increased very significantly compared with group A (p<0.01) . [Conclusion] Combination of Pg-LPS and stress can develop apoptosis of macrophage.Part 4 Effect of Pg-LPS on expression of protein P53 of macrophage and activity of DNA binding in stress rats[Objective]Study of the activity of P53 and protein phosphorylation of macrophages treated with different methods, in order to further conform the internal mechanism that Pg-LPS and stress can bring diseases. [Methods] 12 male SD rats with SPF grade were used in this study. They were randomized into 4 groups, i.e. group A (normal control group), group B (stress group), group C (Pg-LPS group) and group D(stress+ Pg-LPS group). Abdominal macrophages were extracted respectively, then EMS A was used to detect the protein P53 and activity of DNA binding and Western blot was used to detect expression of P53 and the phosphorylation site (Serl5 and Ser392). [Results] (1) Protein P53 and activity of DNA binding: group D increased significantly compared with group A (P<0.05 ) and no difference was found between other groups (p>0.05) . (2) Western blot: ? There is no significant difference of total protein of P53 between each group (p>0.05). (2)P53-Ser15 in group D increased significantly compared with group A (P<0.05) and no difference was found between other groups (p>0.05) . (3)P53 —Ser392 in group D increased significantly compared with group A(P<0.05 ) and no difference was found between other groups (p >0.05 ). [Conclusion] Combination of stress and Pg-LPS take significant roles in phosphorylation of P53 and increase of the activity of DNA binding of protein P53.
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