| Inflammation, the focus of international medical field, is closely related to various human diseases. It remains the fundamental topic in the basic research and clinical practice. Research has showed excessive inflammatory responses constitute the pathological basis of the multiple post-traumatic complications, such as sepsis/septic shock, acute respiratory distress syndrome and multiple organ dysfunction syndrome. So we have studied the mechanism and key controlling points in inflammatory responses from the membrane receptors in effector cells, intracellular signaling pathway expression and regulation of targeting gene, apoptosis of the effector cells, seeking anti-inflammatory measures. In the past five years, we have not only made progress in disclosing the mechanism of post-traumatic inflammatory responses, but also found that the self-protecting anti-inflammatory effects did influence the occurrence and progress of inflammation. In addition, research has demonstrated that the monocytes may secret more pro-inflammatory mediators in the early phase of severe injury, which is inhibited with time prolonged. The secretion of anti-inflammatory mediators, such as IL-1Ra, IL-10, has no obviously attenuation. In view of the above results, we speculate inflammation is a complicated course of mutual promotion and restraint between pro-and anti-inflammatory response. Although endogenous anti-inflammatory effects maintain the homeostasis, the disturbance of the mechanism of anti-inflammatory responses, not only promotes the transformation of inflammation from protecting stage to destructive outcome, but also results in the hypo-immunity of the injuring bodies, which is the reason for high sensitivity to infection. However, little is known about the self-regulating mechanism. In recent years, basic and clinical studies have demonstrated peroxisome proliferator -activated receptor γ(PPARγ), belonging to the nuclear steroid receptor superfamily, suppresses the pro-inflammatory responses by transaction. PPARγis widely distributed in macrophages, dendritic cells, T cells, B cells and others. Previous researches have showed it plays an important role in lipid metabolism and glucose homeostasis. Recently, PPARγis also found to be the checkpoint in controlling inflammation. Research on the anti-inflammatory effects of PPARγhas showed that its agonists obviously inhibit the secretion of pro-inflammatory mediators such as TNFα, IL1, IL6 and NO, in activated monocytes and macrophages. Pretreatment of wild-type mice with PPARγligands can decrease the expression of pro-inflammatory cytokines, alleviate the injury of local and distant tissues, which plays the therapeutic effect on many inflammatory diseases, such as acute myocarditis, autoimmune encephalitis and multiple sclerosis. Unfortunately, PPARγgene knockout have resulted in embryonic lethality because of placental dysfunction. Heterozygous mice (PPARγ+/-) used to investigate the role of PPARγshowed some limitation because lower levels of PPARγremains regulating diverse cellular processes in cells. On account of its distinctive action, transacting various transcription factors and/or cytoplasmic co-factors, we think that PPARγmodulates not only the synthesis of pro-inflammatory mediators, but also the production of anti-inflammatory mediators, which is the key checkpoint in keeping the balance of pro-and anti-inflammatory responses. RNA interference (RNAi), a powerful technique for selectively silencing the expression of genes, is firstly discovered in threadworms by Fire et al, followed by the detection in fruit fly, insects, plants and mammals. In RNAi pathway, double-stranded RNA (dsRNA) is cleaved into fragments of 21-25 nt, which degrades the sequence-specific mRNA facing the target mRNA of cognate complementary sequences, causing the post-transcriptional gene silencing (PTGS). Experimental cells show the phenotype of specific gene deletion. As an operative simple technique instead of gene knockout, it is easy to operate and economical. Compared to the antisense technology, the suppression on the targeting genes is more specific and more efficient. So research on the candidate gene function and gene therapy by RNAi technique has become the hot spot in life science nowadays. In view of the behavior of PPARγon various key transcriptional factors, this research firstly investigates the dynamic expression of PPARγ, then, constructs the recombinant pSUPER.EGFP vectors including the inserts of PPARγtargeting sequences. Finally, observes the silencing effect of PPARγsiRNA on the macrophage cell line RAW264.7. On this basis, we further studied the mechanism of PPARγon inflammatory response, especially that of signal transduction of cellular anti-inflammatory effect, in order to further elucidate the role of PPARγin controlling the dynamic equilibrium between pro-and anti-inflammatory responses, and disclose the mechanism of self anti-inflammation in orderto supply a new idea for research on inflammation. The main results are as follows, 1. LPS up-regulates the PPARγexpression of macrophage cell line RAW264.7, mainly the cytoplasmic part, in a time-dependant manner. The changes of PPARγin the nucleus is less evident expect the down-regulation in 16 hours of LPS stimulation. During the course of observation, the protein ratio of cytoplasmic PPARγto nuclear PPARγshows the tendency of up-regulation. 2. Curcumin stimulates the expression of cytoplasmic PPARγprotein drastically in RAW264.7 cells, while the nuclear PPARγpart is also up-regulated. The protein ratio of cytoplasmic PPARγto nuclear PPARγshows the tendency of up-regulation. 3. After pretreatment of RAW264.7 cells with curcumin, LPS stimulation has increased the amount of PPARγprotein either in the cytoplasm or in the nucleus. Also, the nuclear PPARγprotein is more than that of LPS group in the time point of 12th,16th and 24th hour. The protein ratio of cytoplasmic PPARγto nuclear PPARγalso shows the tendency of up-regulation. 4. In constructing the recombinant plasmids for RNA interference, four kinds of PPARγ-pSUPER-EGFP have been constructed successively by optimizing the selection of targeting sequences and the condition of construction, which knock down the PPARγexpression at the level of transcription and translation in RAW264.7 cells differently. Among them, PPARγ-pSUPER-EGFP2 is most efficient. 5. The expression of phosphor-p42/44 MAPK increased drastically after LPS stimulation. The PPARγgene silencing has no effect on this course. However, the expression of IkappaB-alpha attenuated dramatically after LPS stimulation, while the PPARγgene silencing further strengthened the degradation of IkappaB-alpha. After PPARγgene silencing, curcumin have inhibited the degradation of IkappaB-alpha to some extent following LPS stimulation. 6. The level of TNFαand IL-1Ra increased after LPS stimulation. The peak of TNFαis ahead of that of IL-1Ra. Curcumin has decreased the TNFαlevel. After PPARγgene silencing, the inhibition of curcumin on TNFαexpression becomes mild. Different doses of rosiglitazone have inhibited the TNFαexpression with or without the PPARγgene silencing. 7. Curcumin has no significant effect on the IL-1Ra level in RAW264.7 cells after LPSstimulation. No difference is found between pre-and post-gene silencing of PPARγ. Different doses of rosiglitazone have increased the IL-1Ra level. After 8 hours of LPS stimulation, the expression of IL-1Ra is more than that of control group, but PPARγgene silencing has no effect on this course. Conclusions 1. The PPARγexpression of macrophage cell line RAW264.7 is mainly located in the cytoplasm. Curcumin not only has up-regulated its expression and accelerated the translocation of PPARγprotein from cytoplasm to nucleus, but also reversed the inhibitory effect of translocation of PPARγprotein from cytoplasm to nucleus induced by LPS. The modulation of the PPARγexpression and its nuclear-cytoplasmic shuttling may be the key point of controlling the inflammation. 2. In constructing the recombinant plasmids for RNA interference, the optimization of the selection of targeting sequences and the condition of construction serves the plasmid-induced RNAi research. The efficient PPARγ-pSUPER-EGFP not only establishes the basis for the construction of PPARγdeficient cell line, but also supplies the cell model for the research on the PPARγgene function and gene therapy in corresponding diseases. 3. The anti-inflammatory responses induced by PPARγdoesn't change the activation of ERK signaling, but may attenuate the phosphorylation and degradation of IkappaB-alpha, and inhibit the activation and translocation of NF-κB. The PPAR γdependant anti-inflammatory responses induced by curcumin includes the antagonism on NF-κB signaling. Also, PPARγmay not be the only effector molecule. Other signal molecules in the downstream probably plays an anti-inflammatory role in a PPARγindependent manner. PPARγbelongs to one of the targeting molecules when curcumin works. 4. The pro-and anti-inflammatory responses maybe works consequently in RAW264.7 cells. The PPARγexpression induced by curcumin is negatively related to the TNFαlevel after LPS stimulation, but has no relationship with the IL-1Ra level. The PPARγligand rosiglitazone stimulates the expression of IL-1Ra in a PPARγindependent manner. The synergistic anti-inflammatory strategy with curcumin and rosiglitazone may be more efficient in controlling the macrophage inflammation.
|
PDF Full Text Request