The Study On Regulation Mechanism Of JNK1 And TIPE2 For Diaphragm Weakness In Sepsis

IntroductionDiaphragm is important respiratory muscles of the body, representing 60%~80% for the maintenance of an organism’s respiratory function has an extremely important role. Studies have found that infection can cause diaphragmatic muscle weakness of the body, but the mechanism is not clear. There are many reasons for infetion, such as chest infections, sepsis and septic shock, we mainly study of endotoxemia in the rat diaphragm dysfunction related mechanism.According to the results of our team previous research before, we found that JNK1 signal pathway plays an important role in the course of diaphragmatic muscle weakness, accordingly this experiment using lipopolysaccharide act on SD rats, building diaphragmatic muscle weakness model, research the diaphragm muscle function obstacles of related proteins changes in endotoxemic rats, while we also injection with ip SP600125 a JNK1 inhibitors. After JNK1 pathway was inhibited, observation SD rats diaphragm muscle of related function whether get protection and the function of diaphragm muscle contractility was restored. TIPE2 as an immune regulatory factors, plays a very important role in the process of inflammation, this experiment did to TIPE2 gene detection in order to clarify JNK1 pathway related proteins and the role of TIPE2 gene in diaphragmatic muscle weakness. ObjectiveConstruction endotoxemia rats diaphragmatic muscle weakness model, study JNK1 pathway related proteins and the role of TIPE2 gene in diaphragmatic muscle weakness, at the same time C2C12 LPS infection model constructed to futher clarify the regulation of JNK1 pathway. MethodsForty rats, weight 180~220g were random divided into three groups: normal control group, LPS group and LPS +SP600125 group. The rats in LPS group were injected with LPS at 12mg/kg intraperitoneally. The rats in LPS +SP600125 group were received SP600125 at 30μM/kg at the same time also injected with LPS at 12mg/kg intraperitoneally. Contol group injected with same dose saline. All groups were also injected subcutaneously with 30 ml/kg saline. At 24 h after injections, animals were anesthetized with pentobarbital the parameters of respiratory function and the artery blood gas analysis were also detected. Diaphragm strips were isolated to observe peak twitch tension(Pt), maximum titanic tension(Po), time to peak contraction(CT), half relaxation time(1/2RT), maximal rate of contraction(+d T/dtmax), maximal rate of relaxation(-d T/dtmax), force-frequency curve and fatigue index(FI). Meanwhile, collecting diaphragm specimens for Western Bolt and RT-PCR experiments, detect the gene and protein expression. Using HE-staining and immunohistochemical methods observed morphological changes of diaphragm. Build JNK1, TIPE2 overexpression and silencing plasmid, plasmid transfection into mouse C2C12 skeletal muscle cell model using Western Blot and Real time-PCR to further explore JNK1 pathway, mechanism of TIPE2 in diaphragm muscle weakness. Results(1)Compared with control group, RF, TV, PV and MVV in LPS group were significantly decreased(P ﹤ 0.01). LPS+SP600125 goup versus with LPS group the values were significicant increased(P﹤0.01).(2) Pt, Po and ±d T/dtmax in LPS group were lower than those in control group(P﹤0.01), CT, 1/2RT were higher than those in control group(P﹤0.01). Pt, Po and ±d T/dtmax in LPS+SP600125 group were hiher than those in LPS group(P﹤0.01), CT, 1/2RT were lower than those in LPS group(P﹤0.01).The above six indexes in LPS+SP600125 group did no difference in contrast to control group(P>0.05).(3) Western Blot displayed TIPE2, and p-JNK of expression volume obviously increased in LPS group, and JNK1 no obviously changes, after inhibiting the expression of JNK1, p-JNK in low expression in LPS+SP600125 causes TIPE2 rendering obviously of declined trend of expression also, and RT-PCR get consistent results.(4) Light microscopy observe HE-stainingshowed that control group rats diaphragm muscle fiber shape more rules, arranged neatly, but myofibrils were swollen and mitochondria were vacuolization in LPS group.(5)Immunohistochemical results show TIPE2 expression in LPS group was significantly higher than the Control Group, the darker, the difference was statistically significant(P<0.05), JNK1 test results showed no significant change.(6)C2C12 muscle cell model shown at different points, p-JNK and TIPE2, with increased expression of LPS stimulation time, 24 h after the handle. Then we constructed JNK1 and TIPE2 overexpression vectors transfected into C2C12 cells and detected by RT-PCR method, which shows a positive correlation between the expression of this consistent with the animal model of experimental results. Conclusions1. Endotoxemia rats showed diaphragmatic weakness, diaphragmatic function improved after SP60125 injected indicates that SP600125 may have some protective effect on diaphragm.2. JNK1 pathway can be activated after LPS stimulation in rats, p-JNK, TIPE2-expression were increased. After JNK1 were inhibited, the expression of p-JNK and TIPE2 were decreased and the diaphragm muscle contractility recovery in some extent indicates that changes in rat diaphragm muscle contractility may be related to JNK1 pathway regulation.3. There is a positive regulation relationship between TIPE2 and JNK1.