Roles Of P38Signal Transduction Pathway In Rat Corneal Neovascularization

BackgroundThe cornea locates in the front of the eyeball which has much refractive power and the special transparent and flexible structure without vessels. A normal cornea has five layers:epithelium layer, Bowman’s layer, stroma, Descemet layer and endothelial layer. The epithelium layer is composed of several layers of epithelial cells; Corneal cells can be found in the stroma layer; The endothelial layer is consist of monolayer of endothelial cells. Its transparency is due to its uniform structure, avascularity, and deturgescence. The corneal limbus has lots of vascular networks which can transport nutrition to peripheral cornea. Of course, aqueous and tears are also the nutrition resources of cornea.The peripheral vascular networks of the corneal limbus will expand under the conditions of inflammations, trauma, immunity and infections. Then new vessels gradually invade into the transparent part of the cornea and corneal neovascularization (CNV) will begin to accumulate. CNV can promote inflammation absorption, corneal recovery and improve blood supply. At the same time, CNV may destroy the mechanism of the immune privilege, and reduce the corneal transparency. CNV is a great challenge for further corneal treatment.It is of great importance to further the study of the mechanism and the growth of CNV. Many factors contribute to CNV. Among these factrs inflammation plays an important role. CNV and inflammation promotes, depend on, and collaborate each other. Inflammatory cells invade the cornea before the CNV appears. The position of inflammatory infiltration agrees with that of CNV. CNV appears because the balance between angiogenic growth factors and inhibitory factors has been destroyed by the inflammatory responce.The research of signal transduction is the hot spot and frontier in the study of life science, and the basic research ideas have already catched the eyes of the scientists’. The research of signal transduction may provide a new strategy to explain the life phenomenon, cell metabolism, tumor molecular mechanism and pathological reaction process. Mitogen-activated protein kinase (MAPK) which is composed by a series of serine and threonine can be stimulated by extracellular irritation. MAPK can phosphorylate other proteins in cytoplasm and transfer into nucleus. Many transcription factors may be stimulate by the actived MAPK, then complicated biological functions come into being. Highly conservation MAPKs in mammals may be important messangers which convey stimulates from outsides into nucleus and may be of great importance in embryonic development, cell differentiation, cell proliferation, cell death, cell adaption and inflammatory responces.At present, four types of MAPKs have been found and they are ERK, c-Jun, p38and ERK5respectively. ERK may play a big role in cell differentiation, cell transformation and cell proliferation; c-Jun may participate in embryonic development, cell differentiation and tumorigenesis; ERK5may take part in cell cycle, cell proliferation and regulation of gene expressions; and p38signal transduction pathway is considered to be connected with inflammatory responces.The activation of p38signal transduction pathway is the final step of phosphorylation cascade. The phosphorylation of p38(p-p38) is the activated form of p38. p-p38can enter into nucleus immediately and stimulate many protein kinases and transcription factors. It is of great importance for p38signal transduction pathway in inflammation regulation because of its efficiency and multiple effects. p38signal transduction pathway may draw more attention away and may be a new strategy to study the inflammatory correlation disease.Inflammation has close ties with CNV, and p38signal transduction pathway has an important role in the regulation of the inflammatory response. So we could infer that p38signal transduction pathway might have the possible ability to regulate the growth of CNV. Further studies indicated that inhibitors of p38pathway had anti-inflammatory ablility and SB220025which was one of the p38inhibitors could reduce vascular density in granuloma tissue. The mechanisms of CNV were still unknown. p38signal transduction pathway might be a new target to explore the mechanisms of CNV due to its complicated functions in the progress of inflammation.In our study, p-p38expressions in rat corneas would be detected in order to discuss the relationship between p38pathway and CNV; The toxic effect of SB203580which is a special inhibitor of p38on rat corneas would be dbserved in order to assess the possibility to further study; The inhibitory effects of SB203580on rat CNV would be discovered in order to analyze the roles of p38; At last soluble vascular endothelial growth factor receptor2(sVEGFR2) would be used to explore the relationship between the p38pathway and the VEGF pathway.Part I Expression of p-p38in Progress of CNV in RatsObjectiveExpressions of p38and p-p38were detected in the progress of CNV in rat models. To analyze whether the growth of CNV was relevant to p38signal transduction pathway; To study the time and space distribution of p38and p-p38expressions; In order to confirm CNV is relevant to p38pathway and early activation of p38may be one of the key factors which promote CNV.Methods1. CNV Models Suture-induced CNV was produced in SD rats. Only the right eye was chosen. Three intertupted sutures which hit into the corneal stroma were carried out at the position of lmm inside the limbus. The rats after operations were randomly divided into three groups:the first postoperative day group, the third postoperative day group and the seventh postoperative day group. Normal corneas without sutures were selected as the negative control group. Every group had eight SD rats.2. Clinical Observation Rat corneas were observed by the slit lamp microscope. Pay close attention to whether rat corneas are transparent and complete; to whether sutures are loose; and to whether there are corneal infections.3. Area of CNV Robert formula was used to assess CNV area according to the different group. The length of CNV was recorded through the operating microscope.4. Expressions of p38and p-p38in Progress of CNV On the first, third and seventh postoperative day, five rats from every group were randomly chosen and corneas were harvested for Western Blot detection. The negative control group received the same procedure. The ratio of the IOD of the target proteins to that of GAPDH was defined as the relative expression.5. Immunofluorescent Staining At the specified time corneas were harvested for staining according to the groups. Expressions of p38and p-p38were detected.6. Statistical Analysis All the measurement datas were recorded by mean±standard deviation. Statistical analysis was carried on through SPSS13.0. One-way ANOVA was used to compare means when homogeneity of variance was confirmed and Welch was appropriate when homogeneity of variance was not confirmed. For multiple comparisons, LSD was chosen in the former condition; Dunnett T3was chosen in the later. Two-side test was used for statistical inference. The test level was0.05.Results1. Clinical Observation Vascular network near the limbus would begin with congestion and cornea near sutures would become thick on the first postoperative day. Heavy ciliary congestion and new slim corneal vessels would be found on the third postoperative day. On the seventh postoperative day, plenty of CNV would be observed, and CNV would invade through the position of sutures. The cornea would lose its part transparency.2. Area of CNV No new vessels were found in the corneas on the1st postoperative day and normal corneas had no vessels. The area of the3rd and7th postoperative day group was4.19±1.22and7.26±0.72mm2.3. Results of Western BlotThe relative expression of p38was0.5681±0.1074in the normal cornea group;0.5512±0.0795in the first postoperative day group;0.5517±0.0908in the third postoperative day group; and0.5641±0.1168in the seventh postoperative day group. No significant difference was found among the four groups (F=0.037, P=0.990.).The relative expression of p-p38was0.2969±0.0475in normal cornea group;0.6219±0.0609in the first postoperative day group;0.2812±0.0602in the third postoperative day group; and0.3149±0.0496in the seventh postoperative day group. Significant difference was found among the four groups (F=43.895, P=0.000). The relative expression of p-p38in the first postopertative day group was much higher than that in the other groups; No significant difference of p-p38expression was found in normal cornea group, the third postoperative day group and the seventh postoperative day group.4. Results of Immunofluorescent StainingExpressions of p38were found in inflammatory cells, endothelial cells of CNV and epithelial cells. p38was located in cytoplasm. No obvious difference was found among these four groups.Expressions of p-p38were found in inflammatory cells and endothelial cells. p-p38was located in nucleus. p-p38expressed the most strongly in the first postoperative day group than in the other three groups.Conclusion1. In the progress of CNV, no obvious change of p38expression was discovered, but p-p38expression had reached an all-time high in the progress of CNV on the first postoperative day.2. After suture methods, there was activation of p38signal transduction pathway first, then CNV appeared.3. CNV might be related to activation of p38signal transduction pathway.4. Early activation of p38pathway might be an initiation factor which promoted CNV in suture-induced models.Part II Toxicological Effect of50μmol/L SB203580on rat corneas via subconjunctival injectionObjectiveTo study the toxicological effect of SB203580with subconjunctival injection on rat corneas; To search the appropriate concentration of SB203580; And to provide the basis for further studies.Methods 1. Preparation of50μmol/L SB203580lmg SB203580was dissolved in0.05ml cold dimethyl sulfoxide, then the mixture was diluted into53ml cold normal saline. The solution was kept at the temperature of-20℃.2. Grouping Method After1week’s adaptive feeding SD rats were randomly divided into two groups:50μmol/L SB203580group and normal saline group. Each group had ten rats.3. Subconjunctival Injection Only the right eyes were chosen for research. The injection site was0.5-1mm outside the corneal limbus. Usually the order is from zero to twelve, clockwise. The injections were carried out daily for7days with normal saline or50μmol/L SB203580according to the group. The injection volume was0.04ml.4. Clinical Observation and Corneal Inflammation Index Corneas were examined daily through a slit lamp microscope. Pay close attention to the conjunctival hyperemia, corneal transparency and the integrity of the epithelium. On the seventh day after injections corneal inflammation index was recorded by our evaluating central corneal edema, peripheral corneal edema and ciliary congestion.5. Histopathological and Ultrastructural Examination Corneas were harvested after seven days’ injection for examination. One half was fixed by polyphosphate formaldehyde for histopathological examination, and the other half was fixed by glutaraldehyde for transmission electron microscopy examination.6. All the measurement datas were recorded by mean±standard deviation. Statistical analysis was carried on through SPSS13.0. t-test was used to compare means. Two-side test was used for statistical inference. The test level was0.05.Results1. Clinical Finding Reversible conjunctival anemia was found in50μmol/L SB203580group and disappeared on the following day. Corneal transparency and integrity was kept in both groups in the observation period.2. Corneal Inflammation Index Following7days’ injection the index of the SB203580group was0.14±0.07, and that of normal saline was0.21±0.10. No significant difference was found in the two groups (t=1.716, P=0.103). The ciliary congestion score of the SB203580group was0.70±0.48, and that of normal saline was1.30±0.67. Significant difference was spotted between the two groups, and the ciliary congestion score of normal saline group was higher than that of SB203580group(t=2.286, P=0.035).3. Results of Histopathological and Ultrastructural Examination Histological examination revealed that all rat corneal epithelium were completed, the stroma was arranged in order and the cortex was continuous. No obvious abnormal corneal structures and organelles were found under transmission electron microscope.Conclusion1. Subconjunctival injection of50μmol/L SB203580had no obvious toxicological effect on rat corneas.2. The ciliary congestion score of normal saline group was higher than that of SB203580group. We inferred that this might be related to the anti-inflammatory ability of SB203580.Part III Inhibitory Effect of50μmol/L SB203580on CNV via Subconjunctival InjectionObjectiveTo study the inhibitory effect of SB203580on CNV with subconjunctival injection in rats; To detect expressions of p-p38and VEGFA in CNV models after injections of SB203580; To explore the possible mechanisms of its inhibition.Methods 1. CNV Models Suture-induced CNV was produced in SD rats. The rats after operations were randomly divided into two groups:group with subconjunctival injection of50μmol/L SB203580and group with subconjunctival injection of normal saline. Each group had eight rats.2. Subconjunctival Injection The injections were carried out daily for7days with normal saline or50μmol/L SB203580. The injection volume was0.04ml.3. Clinical Observation Corneas were examined daily through a slit lamp microscope. Pay close attention to the conjunctival hyperemia, corneal transparency, epithelial integrity and corneal infection.4. Area of CNV On the seventh postoperative day Robert formula was used to assess CNV area. The length of CNV was recorded through the operating microscope.5. Expressions of p-p38and VEGFA On the seventh postoperative day, five rats from each group were randomly chosen and the corneas were harvested for Western Blot detection.6. Histopathological and Immunohistochemical Examination On the seventh day after operation three rat corneas from each group were harvested for HE staining and immunohistochemical staining of CD31.7. All the measurement datas were recorded by mean±standard deviation. Statistical analysis was carried on through SPSS13.0. t-est was used to compare means. Two-side test was used for statistical inference. The test level was0.05.Results1. Clinical Observation In normal saline group, vascular network near the limbus began with congestion on the first postoperative day; Heavy ciliary congestion and new slim corneal vessels would be found on the third day; On the seventh day, plenty of CNV would be observed, and CNV would invade past the position of sutures. In SB203580group, on the first day no obvious congestion of vascular network was found; On the third day, few slim new vessels appeared; On the seventh day, CNV could not reach the position of sutures.2. Area of CNV On the seventh postoperative day, the area of SB203580group was4.78±1.25mm2and that of normal saline group was6.86±1.25mm2. The area of SB203580group was less than that of normal saline group (t=-3.319, P=0.005). Subconjunctival injections of50μmol/L SB203580could inhibit the growth of CNV in rat models.3. Results of Western BlotOn the seventh day the relative expression of p-p38in SB203580group was0.2603±0.0396, and that in normal saline group was0.3937±0.1039. Relative expression of p-p38in SB203580group was much less than that in normal saline group (t=-2.682, P=0.028).The relative expression of VEGFA in SB203580group was0.6904±0.1032, and that in normal saline group was0.7411±0.1007. No significant difference was found between the two groups (t=-0.786, P=0.454).Subconjunctival injection of50μmol/L SB203580could inhibit the expression of p-p38, but could not inhibit VEGFA expression.4. Results of Histopathological and Immunohistochemical ExaminationOn the seventh day after operation, corneal tissues were arranged in order, the lumen of CNV was tiny, the density of CNV was low, and inflammatory cells infiltrated less in SB203580group. The high expression of CD31was spotted in the endothelial cells of CNV.In normal saline group, lots of vessel-like structures were observed, the lumen of CNV was more heavy than that in SB203580group, the inflammatory cells infiltrated seriously and irregularly. Further CD31staining revealed that positive cells located in CNV and inflammatory cells intricately.Conclusion1.50μmol/L SB203580via subconjunctival injections could inhibit the growth of CNV in rat models.2. Subconjunctival injections of50μmol/L SB203580could inhibit the expression of p-p38, and could not inhibit VEGFA expression.3. Subconjunctival injections of50μmol/L SB203580might inhibit inflammatory responces.4. p38signal transduction pathway might be related to the regulation of CNV. Activated p38pathway could promote CNV and the p38inhibitor could depress CNV.5. Targeting blockage of p38signal transduction pathway might be a new strategy for CNV therapy.Part IV Inhibitory Effect of sVEGFR2on p-p38in progress of CNVObjectiveTo study the inhibitory effect of sVEGFR2on CNV by targeting blockage VEGF and its receptors; To explore the roles of sVEGFR2in activation of p38signal transduction pathway; And to analyze whether VEGF participated in p38regulation on CNV.Methods1. Rat models Suture-induced CNV was produced in SD rats. The rats after operations were randomly divided into two groups:group with subconjunctival injection of sVEGFR2and group with subconjunctival injection of normal saline. Each group had eight rats.2. Subconjunctival Injection The injections were carried out daily for7days with normal saline or sVEGFR2. The injection volume was0.04ml. 3. Clinical Observation Corneas were examined daily through a slit lamp microscope. Pay close attention to corneal transparency, epithelial integrity, corneal infection and CNV.4. Area of CNV On the seventh postoperative day Robert formula was used to assess CNV area. The length of CNV was recorded through the operating microscope.5. Expressions of p38and p-p38On the seventh postoperative day, five rats from each group were randomly chosen and the corneas were harvested for Western Blot detection.6. HE and Immunofluorescent Staining On the seventh day after operation three rat corneas from each group were harvested for HE staining and Immunofluorescen staining of p38and p-p38.7. All the measurement datas were recorded by mean±standard deviation. Statistical analysis was carried on through SPSS13.0. t-test was used to compare means. Two-side test was used for statistical inference. The test level was0.05.Results1. Clinical ObservationIn normal saline group, vascular network near the limbus began with congestion rapidly. On the seventh day, plenty of CNV would be observed, and CNV would invade past the position of sutures.In sVEGFR2group, CNV expanded slowly. On the seventh day, CNV could not reach the position of sutures.2. Area of CNV On the seventh postoperative day, the area of sVEGFR2group was3.87±0.73mm2and that of normal saline group was6.94±0.92mm2. The area of sVEGFR2group was less than that of normal saline group (t=-7.343, P=0.000). Subconjunctival injections of sVEGFR2could inhibit the growth of CNV in rat models.3. Results of Western BlotOn the seventh day the relative expression of p38in sVEGFR2group was0.6179±0.1334, and that in normal saline group was0.6038±0.1484. No significant difference was found between the two groups (t=0.158, P=0.878).The relative expression of p-p38in sVEGFR2group was0.2667±0.0642, and that in normal saline group was0.4771±0.0.1171. The relative expression of p-p38in sVEGFR2group was much less than that in normal saline group(t=-3.523, P=0.008).4. Results of HE and Immunofluorescent StainingOn the seventh day after operation, corneal tissues were arranged in order, the lumen of CNV was tiny and the density of CNV was low in sVEGFR2group.In normal saline group, lots of vessel-like structures were observed. The lumen of CNV was more heavy than that in sVEGFR2group and the inflammatory cells infiltrated seriously and irregularly.No obvious difference of p38immunofluorescent staining was observed between the two groups, but p-p38strongly expressed in nucleus in normal saline group.Conclusion1. sVEGFR2via subconjunctival injections could inhibit the growth of CNV in rat models.2. Subconjunctival injections of sVEGFER2could inhibit the expression of p-p38and surpress the activation of p38signal transduction pathway.3. The relationship between p38signal transduction pathway and VEGF mediated pathway was not only collaborative and interconnected but also independent and self-governed. In the complicated signal network, p38and VEGF might not be arranged in a simple straight line.