The Research Of The Action And Mechanism Of TPCA-1 In Lipopolysaccharide Inducing The Corneal Stromal Fibroblasts

The character of Infectious corneal ulcer is the infiltration of a variety of immune cells in corneal stroma, including neutrofils, macrophages, lymph cells etc. and the action has close relation with many kinds of cytokine such as IL-6 IL-1 TNF-αIL-8 secreted by corneal inherent cells. all these cytokines induce the activation and chemotactic of immune cells. Corneal stromal fibroblasts expressing glycoprotein on cell surface-intercellular adhesion molecular (ICAM-1) can combine the leucocyte-associated antigen which express on all the immune cells surface . the combination of ICAM-1 can strengthen the immune action and play a key role in infectious corneal diseases. The interaction of corneal stromal fibroblasts and infiltrated immune cells play an important role in the pathogenesis.In the procedure of corneal inflammation of mammals ,the recognition of cell surface receptor TLRs with PAMPs lead to a series of signal activation, and expression of cytokine inflammatory factors and costimulatory molecules in antigen-specific and non-specific acquired immune response activation. Lipopolysaccharide is the components on gram stain negative bacteria cell membrane, and considered to promote the corneal inflammation. corneal stromal fibroblasts can recognize lipopolysaccharide and regulate the expression of auto-immune associated factors. In the acute inflammatoty action by injecting LPS into corneal stroma, the character is the infiltration by neutrophils and other immune cells and secondary corneal ulcer . this action can be strengthened by some protein such as soluble CD14 and LPS-coagulation protein in serum transmitted by nuclear transcription factor (NF-кB) .During cytokine signal transduction, nuclear transcription factors play a key role. Nuclear transcription factor NF-кB is a main way of transcription. In normal state, most NF-кB including p50 and p65 subunit is restricted in cytoplasm by inhibitory protein IB. It can be activated by inducing IB protein phosphorylation or degradation, which releases NF-кB starting nuclear translocation and regulate the target expression. The phosphorylation of IB depends on the mixture of IKK, including IKK-1 and IKK-2 and IKK-3 subunit . The activation of IKK-2 is 20 times than IKK-1, that means IKK-2 play an important role in regulating the activation of NF-кB. Small molecule IKK-2 inhibitor may be applied to treat inflammatory disease, such as asthma and arthritis.We apply human corneal stroma fibroblasts cultured in vitro, to measure the change of human corneal stroma fibroblast secretion under the action of LPS. To dicuss the inhibition in lipopolysaccharide and Signal transduction pathway of IKK-2 receptor blocker, which act in the time of lipopolysaccharide stimulating the corneal stromal fibroblasts to secret inflammation cytokine,chemokine and intercellular adhesion molecule. And we will contrast IKK-2 receptor blocker with dexamethasone in the inhibition degree. By doing this, to verify the substitute and collaborate action of IKK-2 receptor blocker.To mainly study these following questions:①the toxicity of LPS to corneal stroma cells;②the change of corneal stromal fibroblasts secreting inflammation cytokine,chemokine and intercellular adhesion molecule under the action of LPS.③the inhibition of IKK-2 receptor inhibitor (TPCA-1) to corneal stromal fibroblasts secreting inflammation cytokine,chemokine and intercellular adhesion molecule;④the difference of signal transduction ways between IKK-2 receptor inhibitor (TPCA-1) and dexamethasone;⑤the collaboration of IKK-2 receptor inhibitor (TPCA-1) and dexamethasone. Through these studies, to detect the inhibition and signal transduction of corneal stromal fibroblasts to secret inflammation cytokine,chemokine and intercellular adhesion molecule, which is induced by LPS. By doing this, we can provide useful clues for clinical therapy for corneal ulcer .The results show that: 1. on the third day after cells inoculation, cells adherent to the wall, spindle, a round nucleus in center, primary stromal cells may get 80% integration after cultured 3-4 weeks. Afer the first cell passage, cells can merge to grow every 7days to get into next generation . By immunohistochemistry staining we can verify which is the corneal stroma cell. The cells stained by vimentin behaves tawny (positive) in cytoplasm under microscope , and stained by keratin behaves dark blue (negative).2. By effect of LPS under the dense of 100ng/ml in 48 hours, the quantity and shape appears normal and survival rate has no difference with normal, affected over 24 hours by LPS beyond the dense of 1000ng/ml or affected over 12 hours by LPS beyond the dense of 10000ng/ml, the adherent cells decreased obviously, cells shrink, the cellular gap broaden, flowing cells increase in culture medium and survival rate has obvious difference with normal, moreover, the multiplication and survival rate decreases obviously.3. IL-1,TNF-αcan't be detected in negative control group, the IL-6 dense is (83.68±3.21)×10-3 ng/mL, the IL-8 dense is 5.58±0.14ng/mL. By contrasting with negative control group, the human serum group has no significant difference . IL-1,TNF-αcan't be detected in LPS group, the IL-6 dense is 13.99±1.50ng/mL, the IL-8 dense is 43.54±1.20ng/mL, which is higher than negative control group and has significant difference.4. Without LPS, the effect of TPCA-1 of different dense to the base secretion of IL-8 by human corneal stroma fibroblast cell has not significant difference with negative control group. And the secretion of IL-6 has significant difference with negative control, furthermore , the action increases with the TPCA-1 dense and has dense dependency .The lowest dense is 0.1μM, the maximum is 1.0μM. Adding LPS, TPCA-1 of all the denses has significant difference with non-LPS group in effect of human corneal stroma fibroblast cell secreting IL-6,IL-8. and the action depends on dense, increase with the dense growing. The lowest dense is 0.1μM, the maximum is 1.0μM. Without LPS, dexathamesone has significant difference with negative control group in the effect of human corneal stroma fibroblasts secreting IL-6,IL-8. Also the action increases with the dense. The lowest dense is 0.1 nM ,the maximum is 100 nM. After adding LPS, dexathamesone has significant difference with LPS group in the same way. The action increases with the dense. The lowest dense is 0.1 nM ,the maximum is 100 nM.5.Without LPS, TPCA-1 and dexathamesone collaborate to increase the secretion of IL-6(13.3±0.96)×10-3ng/ml, which has significant difference with TPCA-1 or dexathamesone group. and all the three groups have significant difference with negative control group .The secretion of IL-8 is 1.72±0.02ng/ml, which has significant difference with TPCA-1 group or negative , and has not significant difference with dexathamesone group. With LPS, TPCA-1 and dexathamesone collaborate in secretion of IL-6 (0.23±0.02ng/ml). The secretion of IL-8 is 22.41±1.25 ng/ml which has significant difference with dexathamesone or TPCA-1 group.6. The negative group express IL-6 and ICAM-1; the expressive level increases after adding LPS and has significant difference comparing with negative group. Both dexathamesone and TPCA-1 group can inhibit the corneal stroma cells to express IL-6 and ICAM-1. Moreover they collaborate to drop the expressive level furthermore and has significant difference comparing with dexathamesone and TPCA-1 group. Experimental group has significant difference in compare with negative control group.7. The negative group express IL-6,IL-8 and ICAM-1mRNA ,and the expressive level increases after adding LPS, which has significant difference in compare with negative group . Both dexathamesone and TPCA-1 group can inhibit the corneal stroma cells to express IL-6 and ICAM-1 .Moreover they collaborate to drop the expressive level furthermore and has significant difference comparing with dexathamesone and TPCA-1 group. Experimental group has significant difference in compare with negative control group.8. We stain the cell climbing film by immunohistochemistry method. The cell nucleus behaves tawny (positive) in LPS group; and behaves dark blue (negative) in TPCA-1 group; and stained by NF-κB the nucleus express tawny (positive) in dexathamesone group.We can get the following conclusion according the results:1. LPS don't get cell poisoned below the dense of 100ng/ml and act no more than 48 hours, which may be used to study the human corneal stroma cells cultured in condition of inflammation. and the productivity and survival rate of human corneal stroma cells decreases when the dense of LPS increases .2. The cultured human corneal stroma cells don't express IL-1 and TNF-α. the human corneal stroma cells express trace IL-6, IL-8 and ICAM-1 without LPS. after adding LPS for 24hours the level of IL-6, IL-8 and ICAM-1 increases obviously.3.Without LPS, TPCA-1 doesn't inhibit the corneal stroma cells to secret IL-8 and inhibit IL-6 obviously. Adding LPS, TPCA-1 inhibits the human corneal stroma cell to secret IL-6,IL-8 significantly and the action depends on density. Dexathamesone inhibit the corneal stroma cells to secret IL-8 and IL-6, moreover it also inhibit the secretion of IL-8 and IL-6 after adding LPS, which also has dense dependency.4. Both dexathamesone and TPCA-1 group can inhibit the corneal stroma cells to express IL-6; inhibit the corneal stroma cells to express IL-6 and IL-8, moreover they collaborate to drop the expressive level furthermore.5. Both dexathamesone and TPCA-1 group can inhibit the corneal stroma cells to express IL-6, IL-8and ICAM-1 in protein and mRNA level; they drop the expressive level furthermore when collaborate.6. LPS act s on corneal stromal fibroblasts by the signal pathway of activating NF-κB. TPCA-1 can cut down the phosphorylation and degradation of IB and the secondary NF-B nuclear transduction.The corneal ulcer develops rapidly. Besides antibiotics ,new medicine about regulating the function of corneal stromal fibroblasts, inhibiting the degradation of corneal collagen, inhibiting cytokine and chemokine should be exploited and developed . these medicine can cut down the according link to prevent the corneal ulcer. Although dexathamesone can inhibit the expression and increase of a series of chemokine and inflammatory factor of corneal stromal fibroblasts to decrease the expression of MMPs. Dexathamesone has lots of byproduct. So the key problem is the absence of safe medicine clinically. TPCA-1 intervened by LPS can inhibit the corneal stromal fibroblasts to secret inflammatory factor (IL-1, TNF-, IL-6) and chemokine (IL-8) and ICAM-1 on the surface of cell. Compare TPCA-1 with dexathamesone in signal transduction . There is no report of this kind of research, nor the collaboration of TPCA-1 and dexathamesone. Through our research, we suppose TPCA-1 may become the substitute or complementary medicine of dexathamesone in treating infectious corneal diseases.