Role Of TIPE2and PCNP In JNK1Signaling Pathways In C2C12Exposed To LPS

BackgroundSepsis is one of the most common infectious diseases and the major death causer of hospital patients.Patients suffered from severe sepsis take accord1/5of all ones that have to be sent to ICU. What’s more,sepsis makes the main reason of death in non-heart-disease patients in ICU. In America, the death rate ofsevere sepsis is28.6%, amount to215thousand people death from this disease. Sepsis can not only causethe failure of heart, liver, and kidney, but also induce serious and persistent changes in skeletal muscle. Inseptic shock, respiratory muscles were evidently damaged. Septic shock model showed that declinedfunction of respiratory muscles could cause hypercapnic respiratory failure and arrest. The reaction of somefactors lead to respiratory failure, such as endotoxin, inflammatory factor, PG, PAF, reaction oxygen, andNO. The reason for the respiratory failure of patients in ICU is not clear, but most of them suffered frominfection. At least myasthenia of some ones was thought to relate with the function of cytokines[1]. It isshowed that systemic infectious inflammation is the most important reason of secondary diaphragmaticweakness, the mechanism of which is unknown, and few report could be acquired.This study aimed to findthe main signaling pathway of diaphragmatic weakness in infected state, especially the JNK pathway andthe involved target genes and proteins, and use their inhibitors to restraint the decomposition ofdiaphragmatic proteins, which could promote the contractile function of diaphragm and recover forcegeneration fast and effectively.Lots of studies demonstrated that JNK signaling pathway played an important role in celldifferentiation, cell apoptosis, stress reaction and the occurrence and development of many diseases. Thus,JNK signaling pathway is a significant regulation target of cells in both normal and pathological state.Researches confirmed that the main signaling pathways of diaphragmatic weakness caused by infection isp38, JNK1, and PKR, and JNK pathway play a crucial role in the occurrence and development of manydiseases, such as sepsis, COPD, heart failure, uremia, and tumor.The chosen target genes were TIPE2and PCNP. TIPE2is a member of TNF-α induced protein8superfamily, which is a DED protein could regulate apoptosis. It is a key signaling conduction protein relating to cell death and inflammation, and is also a crucial protein that keeping the balance of adaptive and innateimmunity. TIPE2prominently expressed in lymph tissues, and a small amount was also detected in muscletissue. In mice, the absent of TIPE2leaded to multiple organ inflammation, splenomegaly and prematuredeath. TIPE2deficient mice were extraordinarily sensitive to septic shock. TIPE2deficient cells were highresponsive to the activation of TLR and TCR. More importantly, when binding to casepase8, the activationof AP-1and NF-κB was suppressed, apoptosis induced by Fas was promoted. Suppression of casepase-8prominently restricted the high response of TIPE2deficient cells.PEST-containing nuclear protein (PCNP), a nuclear protein which is related to cell cycle regulation,exists in cell nucleus. This novel protein with a RING finger is a ubiquitin-ligases, and relates to tumorgeneration.This study used endotoxin to challenge mouse skeletal muscle cells C2C12, meanwhile, inhibitedJNK1signaling pathway using JNK1-DN, abbreviation of JNK1dominant negative, which had dominantnegative effect. The mechanism was the maturated protein and the wild protein became nonfunctionaldimer, thus, JNK1-DN could inhibit the function of normal genes though competitive inhibition.Researches revealed that inhibitor of JNK1could restrain the activation of casepase3and casepase8, andJNK1had biological relation with TIPE2and PCNP.Nowadays, studies on the therapies of diaphragmatic weakness were explored deeper. These therapieswere targeted to the key molecules which activated JNK signaling pathway. The main signaling pathway ininfectious state, especially the JNK pathway, the molecular biological mechanism of diaphragmaticweakness and the involved target genes and target proteins are the focus of our study.ObjectAddress the molecular biological mechanism of myocytes apoptosis and the involved target genes andtarget proteins through the myocytes infectious models.MethodsMyocytes infectious models was prepared by challenging C2C12cells with endotoxin. MTT assaywas applied to determine the influence of JNK inhibitors on cell proliferation. RT-PCT, semiquantitativePCR was employed to test the influence of JNK inhibitors on the PCNP and TIPE2gene expression. WBwas used to detect the active JNK1,caspase3and caspase8and the surface marker termed myosin. FCM was utilized to observe apoptosis. Meanwhile, PCNP and TIPE2over-expressed recombinant plasmidswere established, which were stably transfected into C2C12cells when the gene sequences were confirmed.After this, relative proteins were successfully expressed in C2C12cells proven by WB. Subsequently, theinfluence of over-expressed proteins on cell proliferation was checked using MTT. The relations ofJNK1,PCNP and TIPE2were tested by WB.Results1. PCNP and TIPE2over-expression recombinant plasmids were constructed, transfected intoC2C12, and PCNP and TIPE2were confirmed to be over expressed in C2C12.2. The stimuli of LPS on C2C12models could activate JNK1pathway and upregulate casepase3and casepase8. JNK-DN inhibited the activation of JNK1pathway, and suppressed the expression ofcasepase3and casepase8.3. JNK1inhibitor upregulated myosin, which is the surface marker of C2C12. Both JNK1inhibitor and PCNP promoted the growth of C2C12cells.4. Over expression of TIPE2upregulated JNK1, while over expression of PCNPdownregulated it.5. No prominent apoptosis was detected when C2C12cells were stimulated by LPS.ConclusionRecombinant plasmids of over-expressed PCNP and TIPE2and muscle cells infectious models wereestablished. JNK1played an important regulatory role in the models. The signaling molecules, such asPCNP and TIPE2affected the cellular functions. These results set an foundation for studies on enhancingthe Contractility of diaphragm, releasing diaphragmatic weakness induced by inflammation and improvingrespiratory function, and then, benefited the critically ill patients who badly needed assisted breathing withventilator.