A Study On Molecular Mechanism In Fibrosis Of Pneumonoconiosis Mediated By TNF-α

Part I The Effects of Oxymatrine and tumor necrosis factor antibody resist TNF-α to induce apoptosis of alveolar macrophageObjective: To study the effects of OM and TNF-α antibody resist the tumor necrosis factor (TNF-α) to induce apoptosis of alveolar macrophage (AM). Materials and methods: AM of the Wistar rats were collected making use of the method of bronchus-lung lavage and the concentration of it was 1×10⁶ml, then to be purify-cultivated and stimulate-cultivated. These cells were divided into four groups: the control group: adding Eagles solution without fetal calf serum; 100μg / ml quartz group: adding Eagles solution without fetal calf serum of containing 100μg/ml quartz; 100μg/ml quartz combined with 10ng/ml TNF-α antibody group: adding Eagles without fetal calf serum of containing 10ng / ml TNF-α antibody at 1 hour before quartz stimulation, then adding 100 μ 1 containing 100 μ g / ml quartz suspending solution; 100 μ g / ml quartz combined with 800 μ g / ml Oxymatrine (OM) : adding Eagles without fetal calf serum of containing 800 μ g /ml Oxymatrine(0M) at 1 hour before quartz stimulation, then adding 100 μ l containing 100 μ g / ml quartz suspending solution. Each group designed four Parallel samples. These cells were cultivated in incubator for 24 hours in 5% CO₂ at 37℃. Then the contents of TNF-α in the cell supernatant solution was detected by the method of enzyme-linked immunosorbentassay (ELISA) and the level of the apoptosis of alveolar macrophage (AM) was detected by the method of TUNEL with fluorescent labelling. Result: TNF-α antibody and Oxymatrine(OM) can degrade the contents of TNF-α in the cell supernatant solution and resist TNF-α to induce apoptosis of macrophage (AM) markedly when TNF-α degrade and which is tested by statistics t-test(P<0.05). Conclusion: 1. Oxymatrine (OM) can resisted silica on AM to secreting TNF-α in dose-dependent manner TNF-α. 2. TNF-α can induced the apoptosis of the AM. TNF-α antibody and Oxymatrine(OM) had markedly effecting for resising TNF-α on the apoptosis of the AM . This experiment may be helpful to further learn the etiopathogenesis of pulmonary fibrosis and the to treat pulmonary fibrosis. Part II The study on the TNF-α mediated signal transductionmechanisms for fibroblasts proliferationObjective: To study the TNF-α mediated signal transduction mechanisms for fibroblasts proliferation , in order to found molecule target spot for the drug designed to treat pneumonoconiosis. Materials and methods: The lung fibroblasts of big neonate rat of the 3 days were primarily cultivated in use of organism clump method and the cells were handed down from generation to generation to 4 - 5 generations when used the cell into this experiment. These good-grow cells were divided into four groups: the control group; 5ng/ml TNF-α group; 10ng / ml TNF-α group; 20ng / ml TNF-α group. Each group designed four Parallel samples. Measuring Indexes: 1.The expression of TNF-α R and PKC on the cellular membrane of fibroblasts was detected by the method of immunohistochemistry. 2. The TNF-α R I (P55) and TNF-α RII (P75) was expressed by the method of Western blot. 3. The level of 1, 4, 5-Trisphosphoinositol (IP3), Diacylglycerol (DAG), Cyclic adenosine monophosphate (cAMP) and Cyclic guanosine monophosphate (cGMP) in cells was studied by the method of radiommunoassay. 4. The contents of Ca⁺² in fibroblasts were detected by the method of flow cytometry. 5. The level of fibroblasts proliferation was detected by the method of 4, 5-dimethylthiazol-2-yl (MTT) .Result: TNF-α can markedly enhance the expression rate of TNF-α R and which has evident statistical significance in comparison with the control. During the different dosage of TNF-α groups, the expression rate of TNF-α R of fibroblasts was the highest in comparison with the rest especially taking the effective dose of TNF-α as 10ng/ml. Because TNF-α R has two kinds: TNF-α R I (P55) and TNF-α R II (P75). In different diseases, the TNF-α coupled receptor were different, and the initiatied signal transduction pathway and the induced biology effect are different. So we made use of the method of Western blot to carrying on the determination of the two kinds in order to further determine which kind of receptor was combined with TNF-α . The results show: TNF-α mainly affects the expression of TNF-α R II (P75) especially in 10ng / ml group, but not have influence to the expression of TNF-α RI(P55). When TNF-α combined with TNF-α RII, IP₃ and DAG which are the important second messengers in cells would raise up following the dosage and the time manner in fibroblasts. IP₃ can induce the release of Ca² in cells. During the different dosage of TNF-α groups, there is the greatest effect to the release of Ca²in cells in 10ng / ml TNF-α group and which is consistent to the expression level of membrane receptor induced by TNF-α and in principle to the expression of IP₃ . the result also explain that the expression level of membrane receptor and IP₃ has related to the release of Ca². The experiment results of DAG and Protein kinase (PKC) showed: the expression level of PKC is unusually consistent to the expression level of DAG and Ca². In order to further confirm the effect to the expression of PKC induced by Ca², we carried out the block-test. Cell were treated with Ca² blocking agent- Nimodipine 1h before using 10ng/ml TNF-α to stimulate cells .We found that the PKC expression level of cell were degraded in comparison with without Ca² blocking agent when Ca² of the cell obviously decrease and there was a statistics difference . The experimental results of cAMP and cGMP show: the level of cAMP and cGMP was various when fibroblasts were effected by different dosage of TNF-α in different time-point. It is interesting that the level of cAMP and cGMP heightened in 5ng / ml and 20ng / ml TNF-α groups following the action-time lasting . The contents of cAMP was heighten markedly at 4h time-point while would trend to decrease when the action -time lasted to 24h at the 10ng / ml TNF-α group, but in opposite the contents of cGMP obviously upgraded from 4h to 24h in this group .hence, the ratio of cAMP/cGMP was obviously lower than the other three groups. The together analysis of Combining with the experimental result of fibroblasts perlioferation experiment, results shows that 10ng / ml TNF-α can enhance the cell peroliferation level and which had obvious statistics difference compared with the other three groups(P<0.05). Conclusion: 1. When combined with TNF-α RII on the membrane of fibroblasts, TNF-α can activate link-coupled PLC and AC kinase with G proteins to open the signal-passageaway which is one of mechanisms to induce fibroblasts peroliferation. 2. the Ca² and DAG co-promote the expression of PKC. The two pathways of IP₃/Ca and DAG/PKC in the signal transduction process may cooperate to induce fibroblasts peroliferation. 3. The ratio of cAMP/cGMP plays important to the peroliferation effect of fibroblasts. When the ratio of cAMP/cGMP heightening, the cell peroliferation would be inhibited and vice versa. 4. 10ng / ml TNF-α play a key role to in the process of fibroblasts peroliferation. PartIII The study of the PGE₂ combined with TNF-α mediated signal transduction mechanism for fibroblasts proliferationObjective: To study the effect of the TNF-α combined with prostanglandin E₂ (PGE₂ ) on signal material of fibroblasts. Research the effect to the proliferation of fibroblasts by the quantity proportion of stimulating factor and inhibition factor to provide the scientific basis for cell factors to treating to pneumonoconiosis. Materials and methods: Big neonate rat of 3days lung fibroblasts were primarily cultured by the method of organism clump and the cell handed down from generation to generation to 4 - 5 generations when used into this experiment. These good-growing cells were divided into 5 groups: 10ng/ml TNF-α, 500pg/ml PGE₂, 1000 pg/ml PGE₂, 2000 pg/ml PGE₂ combined with 10ng/ml TNF-α and control group and each group has four Parallel samples. Indexes: 1. The expression of TNF-α R and PKC on the cellular membrane of fibroblasts was detected by the method of immunohistochemistry. 2. The level of IP₃, DAG, cAMP and cGMP in cells was detected by the method of radioimmunoassay. 3. The contents of Ca² in fibroblasts by the method of flow cytometry. 4. The level of fibroblasts peroliferation was detected by the method of MTT. Result: PGE₂ can inhibit the expression of TNF-α R on membrane of fibroblasts and the expression would be weakened following the increase of PGE₂ .The result was tested by the statistics, P<0.05. The result of IP₃ test shows: when TNF-α combined with 1000pg/mland 2000pg/mlPGE₂, the value of IP₃cpm heightened with the response time increasing and especially obvious in 1000pg/mlPGE₂ group .There was a steep rise from 60s-120s in 1000pg/mlPGE₂ group and highly statistics difference (P<0.01) compared with other four groups. When TNF-α combined with different dosage of PGE₂ and in different response time pionts, the change pattern of IP₃cpm is different. The effect of PGE₂ on Ca²⁺ shows: the Ca²⁺ ion and the expression of PKC in cell at 10ng/ml TNF-α group were compared with the control that were heightened obviously(P<.0.05). When 10ng/ml TNF-α combined with different dosage of PGE₂, the concentration of Ca²⁺ were degraded but compared with the 10ng/ml TNF-α group that there was no statistics difference. The expression of PKC induced by PGE₂ shows: the level of PKC had some rise following the dosage of PGE₂ increasing and there was no statistics difference compared with each group except the control. The result of cAMP and cGMP shows: the expression of cAMP induced by TNF-α combined with different dosage of PGE₂ degraded at the observation time point of 4h but heightened when the response time lasted to 24h and the expression of cGMP degraded with the response time lasting compared with the 10ng/mlTNF-α group. Hence the ratio of cAMP and cGMP heightens at the response time point of 24h following the dosage of PGE₂ increasing. The result of fibroblasts peroliferation experiment shows: the peroliferation effect is most powerful in 10ng/mlTNF-α combined with 1000pg/ml PGE₂ group which the level of cAMP was lower and the contents of IP₃is more than others. Conclusion: 1. 2000pg/ml PGE₂ can obviously enhance the cAMP level of fibroblasts and increase the ratio of cAMP and cGMP and can suppression the peroliferation of the fibroblast, which is related with observation time points. 2. The fibroblast peroliferationis closely related with the proportion of cAMP/cGMP and concentration of IP₃ in the cell. 3. The inhibiting fibrosis effect is mediated by PGE₂.4. The two signal pathways of IP₃/Ca² and DAG/PKC are resisted mutually.Part IV The study of the protect effect of Oxymatrine resistingquartz for Alveolar macrophageObjective: To study the protect effect of Oxymatrine(OM) resisted on quartz forAlveolar macrophage(AM).Materials and methods: AM of Wistar rats were collected in use of the method of bronch lung lavage and the concentration was 1×10⁶, then to be purify-cultured and stimulate-cultured according to the experimental design request. These cells were divided into six groups: 100μg/ml quartz group , 100μg/ml quartz combined with 200 μ g / ml OM , 400μg/ml OM , 800 μ g / ml 0M, 1600 μ g / ml 0M, 3200 μg /ml OM group. Each group has four Parallel samples. The concentration of IL-1 and TNF-α in AM culturing supernatant solution was detected by the method of mouse thymocytes-multiplication and ambi-antibody sandwich methods separately. The vigor of lactate dehydrogenase (LDH) in AM was detected by the method of 2, 4-dinitrophenylhydrazine chromatometry. The lipid peroxidation end- product of malonaldehyde(MDA) in AM was detected by the method of TBA chromatometry. Result: OM can resist on AM to excrete TNF- α and IL-1 induced by quartz. The expression level of TNF-α decreased following the dosage of Oxymatrine (OM) increasing from 200 μ g / ml to 1600 μ g / ml in a dose - dependent manner in the experimental design druing dosage range. The drug action would weaken when the drug dosage was 3200 μg/ml. The expression level of IL-1 also decreased with the dosage of OM increasing especially of rang from 800μg/ml to 1600μg/ml. Conclusion: 1. The OM has the function of anti-oxidation and of protecting the membrane of AM.Part V The study of the proliferation mechanism of the fibroblast by OM combined with TNF-αObjective: The study of the proliferation mechanism of the fibroblast by 0M combined with TNF-α . Materials and methods: Big neonate rat lung fibroblasts were primarily cultured in use of organism clump way for 3 days and the cells were passaged from generation to generation to 4 - 5 generations when used into this experiment then to be purify cultured and stimulate cultured according to the experimental design requests. . These cells were divided into 5 groups: the control, 10ng/ml TNF-α , 200 μ g / ml OM, 400 μ g / ml 0M, 800 μ g / ml OM. Each group had three Parallel samples. Measured Indexes: 1. The expression of TNF-α R and PKC on the cellular membrane of fibroblasts was detected by the method of immunohistochemistry. 2. The level of IP₃, DAG, cAMP and cGMP in cells was detected by the method of radioimmunoassay. 3. The contents of Ca² in fibroblasts by the method of flow cytometry. 4. The level of fibroblasts proliferation was detected by the method of MTT. Results: OM can resist on TNF-α to be combined with receptor and the expression of TNF-α R would decease following the dosage of OM increasing. Meanwhile the expression of PKC, IP₃ and Ca²⁺ would weaken too. The experimental result of cAMP and cGMP shows: the value of cAMP and the ratio of cAMP and cGMP would upgrade gradually with the concentration of OM increasing. When the concentration was 800μg/ml, the value of cAMP and the ratio of cAMP and cGMP was the highest. But there was no statistics difference during the OM groups and was obvious statistics difference during the OM groups and the 10 μ g / ml TNF-α group, P<0.01. The value of cGMP would decrease following the concentration of Oxymatrine(OM) increasing. Conclusion: 1. The OM could resist on TNF-α to induce expression of TNF-α R of fibroblasts, reduced PKC, IP₃. and Ca²⁺ level, rised up ratio of cAMP and cGMP. 2. The mechanism that 0M resist on TNF-α to induce fibroblasts proliferation is mediated by cAMP.