Experimental Study On The Effects Of Ursolic Acid Applied In Rat Corneal Rejection

Immune rejection after corneal transplantation was a complex immune response involved in various immune cells and immune molecules. Though there existed immune privilege and anterior chamber associated immune deviation, rejecting still an important influenced factor to corneal transplanting failure.The prevention and treatment of rejection was still an important topic in the study of corneal transplantation. Currently, nonspecific clinical immunosuppressive therapy are widely used. They were just "superficial" methods, have limited curative effects and more side effects. In recent years, some drugs could be effectively used in corneal graft rejection and prolong corneal graft survival time, which became one of research fields in corneal transplantation rejection.Research BackgroundUrsolic acid (UA), also known as ursolic, WuSu acid, alpha-amyrin, was a kind of weak acid pentacyclic triterpenoid, was a functional component of many natural products. Ursolic acid was a bitter taste, white needle crystal (crystallized in ethanol), and basic frame was more oxygen pinic rings mother nucleus. Chemical name 3-Hydroxy-12-ursen-28-oic acid, molecular formula was C30H48O3, relative molecular weight was 456.68, melting point was 285～291 ℃. It can be easily dissolved in dioxane, pyridine, dissolved in methanol, ethanol, butanol, butanone, slightly dissolved in acetone, benzene, chloroform and ether, undissolved in water and petroleum ether.The study found that UA has obviously cytotoxic effect to a various malignant tumors, induced differentiation and anti-angiogenesis forming and had resistance to different cancer-causing substance and carcinogens. In vitro study shown that it also has resistance in inhibiting tumor cell proliferation and vascular endothelial cells forming.It had various biological effects and pharmacological activities, such as anti-microbial, anti-diabetes, anti-ulcer, hypolipidemic, antioxdant and increased immune function.CD4+Th1 cells played an important role in the process of corneal transplant rejection reaction.Nuclear factor-kappa B was a protein factor extracted in B cell nucleus, combined with a enhancer of kappa B sequence specific of immunoglobulin light chain gene k. It had been confirmed that it widely existed in eukaryotic cells, could specially bind with various cells gene promoter or strengthen subsequence special sites, which promoted transcription and expression. It was closely related to important pathophysiological process such as inflammation, immune response, cell transformation, cell proliferation and apoptosis. Recent studies had found that the nuclear factor-kappa B played an important role in the process of the occurrence and development of various diseases. The nuclear factor-kappa B could increase B7 stimulating molecules and MHC-II on T cell’s surface to express and stimulate Thl cytokines to generate,such as IL-2 and IFN-gamma. Ursolic acid could inhibit activity of nuclear factor-kappa B, thus reducing the expression of IL-2, IFN-gamma molecular and prolonging graft survival time. Immune suppression mediated by ursolic acid was through PKC-Iκ-Bα-NF-κB signaling pathway.UA inhibited I kappa alpha phosphorylation and nuclear factor-kappa Bp65 nuclear transcription, lead to activity of NF-kappa B to be inhibited,which was caused by phosphorylation of PKC. The NF-kappa B was an important regulating factor in the process of immune and inflammatory response,which regulated activity, proliferation and gene transcription of T cells. Heterodimer consisted of p50 and p65,which existed in an non-activated cytosol and combined with inhibiting subunit I kappa alpha-B. Serine of 32 and 36 on inhibiting subunit I kappa B-alpha were phosphorylated through I kappa-B kinase. The purpose of phosphorylation was to ubiquitinate and degradate these units. The degradation of I kappa B-alpha could cause the activating units p50 and p65 to release and combine with specific promoter regions in cell nucleus, which encoded inflammatory cytokines.In the other side, UA could significantly inhibit neovascularizations to come into being and reduced immune rejection inflammatory response; UA reduced expression of CD25 and CD69 and IL-2 on T cell surface,which was induced by CD3 and CD8. UA inhibited transcription of NF-kappa B nuclear factor and activity of T cells, obviously inhibited the NF-kappa Bp65 activation, induced host special immune tolerance in mouse transplantation, rather than raising CD4+CD25+Foxp3+ T cells; UA inhibited phosphorylation of ERK and JNK induced by mitomycin, suppressed activation of immune regulating transcription factor such as NF-kappa B, NF-AT and AP-1, reduced the expression of CD25 and CD69, CD 134, CD80, CD86 in T cell surface, significantly reduced the expression of IL-2, IL-4,IL-6, IL-17, inhibited activation, proliferation and secretion of cytokines of T cells, B cells and macrophages. UA restrained activity of T cells by adjusting NF-kappa B nuclear factor, inhibited activation and proliferation of T cells, reduced the expressions of CD25 and CD69 and CD71 in T cell surface active marker, which were associated with the inhibition of NF-kappa B signaling pathways.Therefore, this experiment intended to apply "ursolic acid inhibits the NF-kappa B to prolong corneal transplantation survival time" to explore that ursolic acid induced corneal transplantation immune tolerance via intraperitoneal injection to allogenic corneal transplantation in rats model (SD�'Wistar) receptors,which could provide important theoretical basis for clinical effective control and treatment to corneal graft rejection reaction. Material and Methods1.Experimental animalsWistar rats were as receptors,while SD rats were as donors. Ages rang from 6 to 8 weeks, weight 180-220g, females, divided in four groups. A:blank control group (n=8),B:autograft control group(n=8),C:allograft control group (n=8),D:ursolic acid group (n=8). Then we established allogenic corneal transplantation model of rats, observed corneal transplantation rejection after operation and scored it according to the criteria rating of Larkin. Obtain materials after 14 days’postoperation.2. Pathological tissue HE StainingMade corneal graft histology tests respectively in the 14th day after operation. After specimens were fixed by 4%paraformaldehyde solution, dehydrated and conventional paraffin embedded, they were cut into 5 microns thickness in serial section.Then histopathological changes were observed by microscope after hemotoxylin eosin (HE) staining.3.Real Time PCR(RT-PCR) assayPut rats cornea in 1 ml Trizol centrifuge tube. Total RNAs were extracted by one-step of Trizol, then tested by agarose gel electrophoresis, RNA concentrations were detected on Nanodrop.Put diethypyrocarbonate (DEPC) water as control, recorded values of OD260/OD280 and RNA concentrations at the same time.Synthesis of cDNA were through reverse transcription reaction by reverse transcription kits. Other operatings were done according to the instructions. DNA samples extracted were directly applied to qPCR experiment or kept at-20℃. Primer sequences as follows:GAPDH Upstream sequence:5’-ACCACAGTCCATGCCATCAC-3’, Downstream sequence:5’-TCCACCACCCTGTTGCTGAT-3’IL-2 Upstream sequence:5’-TGATGGACCTACAGGAGCTCCTGAG-3’ Downstream sequence:5’-GAGTCAAATCCAGAACATGCCGCAG-3’VEGF Upstream sequence:5’-GGCCTCTGAAACCATGAACT-3’ Downstream sequence:5’-TGAACTTCACCACTTGGCAT-3’IFN-y Upstream sequence:5’-GGAGGAACTGGCAAAAGGATGGT-3’ Downstream sequence:5’-TTGGGACAATCTCTTCCCCAC-3’ICAM-1 Upstream sequence:5’-GCTACATCCACACTGACGCT-3’ Downstream sequence.-5’-CAGGGAATGAG TAGACCTCCA-3’NF-κBp65 Upstream sequence:5’-CCGTGGAGTACGACAACATC-3’ Downstream sequence:5’-CCTCTTCCAGCTGCTATGTG-3’RT-PCR reaction system was as follows:the Total volume was 20μl:SYBR@ (R) Primx Ex Taq Ⅱ(Tli RNaseH Plus)(2x)10.0μl、PCR Forward Prime (10μM) 0.8μl、PCR Reverse or word Prime (10μM) 0.8μl、ROX reference Dye (50 x), DNA template 2 μl, dH2O (sterilized distilled water) 6 μl, Pre-degeneration:95 ℃ 30s, reaction conditions:95℃5s,60℃34s,40 cycles. Repeat 3 times.4. Western Blotting assayPut rats corneal graft in frozen RIPA lysaste liquid to crack, PBS buffer washed, then added protease inhibitors to extract nucleoprotein.Proteins were separated by SDS-PAGE gel electrophoresis, transferred to the PDVF membrane, then closed by confining liquid (5%skimmed milk powder) for lh at room temperature;Rabbit polyclonal antibody against NF-kappa Bp65, rabbit anti I kappa B-alpha predominate polyclonal antibody (1:4000), rat monoclonal antibody and anti ICAM-1 were incubated at 4℃ all night.Combined with corresponding IIantibody (1:10000) after being washed and closed, then fixed, developed and exposed. Immune stripes were displayed by chemiluminescence.GAPDH was as reference antibodies. We analyzed immune stripes quantitatively by gel imaging analysis system.5.Statistic method analysisExperimental data were analyzed by SPSS 16.0 statistical software. Average survival time of each group corneal grafts was detected by applying Kaplan-Meier test. Experimental data used x±s to show. We analyzed all the data by one-way anova analysis and two sample mean T test after homogeneity test of variance.If P< 0.05, there was significantly statistical difference.Results1.Compared with rats allogeneic corneal transplantation group, corneal graft survival time in ursolic acid group were obviously prolonged, P< 0.05, there was significantly statistical difference between two groups.2. Each group (normal control group, autologous corneal transplantation, allogeneic corneal transplantation group and ursolic acid group) took eyeballs tissue to make histopathologic slide detection.The results showed that structure of normal cornea tissue was clear, without appearing abnormality. In autologous cornea transplanta-tion group, structure of each layer was clear and no obvious abnormalities,only a small amount of new blood vessels and inflammatory cells infiltrated in stromal layer. In allogeneic corneal transplantation group,a large number of lymphocytes and mononuclear macrophage corneal layers infiltrated in each corneal layer, new blood vessels were visible in stroma,which consisted of lymphoid cells.In ursolic acid group,corneal basic structure maintained normality.Only few lymphocytes infiltrated in stroma, some new blood vessels also existed in corneal graft bed.3. Each group (normal control group, autologous corneal transplantation, allogeneic corneal transplantation group and ursolic acid group)took eyeballs tissue to make Real-Time PCR test. The relative quantity of IL-2, IFN-gamma, the NF-kappa Bp65, VEGF, ICAM-1 had significantly differences at mRNA level in corneal transplantation. The content of IL-2, IFN-gamma, NF-kappa Bp65, VEGF, ICAM-1 increased obviously in group B and group C,while the corresponding inflamma-tion index in group D (namely ursolic acid group) declined obviously after intraperitoneal injection. Applied one-way anova analysis,values of F were 236.991, 414.224,24.931,32.164,21.383. Firstly used homoscedasticity test, then used LSD test to compare each two groups. The content of NF-kappa Bp65 had no significantly statistical difference between group C and group D (P> 0.05),.Except that normal group and autogenous corneal transplantation group had no statistical difference, the rest had statistical differences between groups (P< 0.01).4. Each group (normal control group, autologous corneal transplantation, allogeneic corneal transplantation group and ursolic acid group) took eyeballs tissue to make Western Blotting test. The relative quantity of NF-kappa Bp65 and I kappa B -alpha had differences in corneal graft of each group at the protein expressing level. Autologous corneal transplantation group and ursolic acid group had the similar variation tendency about NF-kappa Bp65 and I kappa B -alpha after corneal transplantation. Compared to allogeneic corneal transplantation group, the relative content of NF-kappa Bp65declined obviously,while I kappa B -alpha increased.in ursolic acid group.Conclusion1.Compared to allogeneic corneal transplantation group, corneal graft survival time of ursolic acid group could obviously prolonged. Pathological tissue HE staining showed that ursolic acid group had less lymphocyte infiltration and new blood vessels.So ursolic acid had certain inhibitory effects on corneal rejection.2.The results of RT-PCR test showed that relative quantity of IL-2, IFN-gamma, NF-kappa Bp65, VEGF, ICAM-1 had differences in corneal graft of each group at the mRNA level. Especially, corresponding inflammation index decreased obviously after using drugs;Western Blotting test results showed that relative content of NF-kappa Bp65 obviously declined, while I kappa B-alpha increased in ursolic acid group.So ursolic acid maybe inhibit corneal transplant rejection by blocking NF-kappa B channel protein expressing.So, this experiment would provide experimental basis for clinical application of ursolic acid.