PIN Involving In Anti-inflammation Effect Of EsA

【Background and Objective】Esculentoside A (EsA) is the major saponin isolated from the Chinese herb Phytolacca esculenta. It has been showed that EsA possessed comprehensive pharmacological effects in the study during the last thirty years. It is supposed that EsA might have potency on treatment of inflammation. The possible mechanisms were investigated through anti-inflammation and immunoregulation after the positive results being found. EsA inhibits over-reactive macrophage by decreasing the expression of NO and down-regulation of phagocytic. The regulatory effects of EsA on inflammation cytokines including IL-1,PGE2,PAF,TNF-a . EsA possess the the pro-apoptosis effect on thymocyte stimulate by ConA. The effect of EsA on gene expression profile changes in cells was investigated using microarray in 2002 by Xiao et al. It was found that EsA could regulate the expression of gene of protein inhibitor of nitric oxide synthase (PIN), tissue inhibitor of metalloproteinase (TIMP), GSK3βand PIAS1. EsA also reconstructs the balance of cytokines by regulation of IL-10, IL-12, TNF-a, TGF-b and regulate the immunological function by increasing the expression of immunophilin (FKBP1a, Cyclophilin).In 1996 Jaffrey and Snyder identified protein inhibitor of nNOS (PIN), in rat brain, which interacts with the NH2-terminal part of the enzyme. Binding of PIN to nNOS prevents homodimerization of the enzyme resulting in an inhibition of NO production. PIN is an ubiquitous protein expressed in rats and humans to an extent depending on the tissue studied. PIN is largely present in testis; moderately in different brain areas, kidney, and ventilary muscles; and faintly in other tissues. Furthermore, PIN has also been identified as the light chain of cytoplasmic and flagellar dyneins and myosin V, two cytoskeletal components involved in the traffic of intracellular organelles and flagellar movements. In addition, PIN is also able to interact with other partners as diverse as Bim (a proapoptotic factor of the Bcl-2 family), IκB (the cytoplasmic inhibitor of nuclear factor-κB), DAP1α(an N-methyl-D-aspartate scaffolding protein), NRF-1 (nuclear respiratory factor-1), Swallow (an mRNA localization protein), and gephyrin (aγ-aminobutyric acid [GABA] or glycine scaffolding protein), suggesting an interplay of PIN in a number of biological functions.Mapping of protein binding interactions with PIN has led to the identification of two consensus motifs, (K/R)XTQT (found primarily in viral proteins that bind PIN) and GIQVD (found primarily in cellular proteins that bind PIN). X-ray diffraction and NMR spectroscopy studies have revealed that these short motifs stabilize interactions with the PIN dimer by binding inside the PIN intermonomer groove, while biochemical studies have confirmed that this conserved motif mediates association between PIN and its protein partners. Additionally, pepscan techniques have demonstrated the ability of short peptides containing these consensus motifs to bind recombinant PIN in vitro. Due to the identification of consensus amino acid motifs that bind PIN, this subunit is considered an attractive target for regulation of PIN-binding protein via a short peptide.Previous studies showed that EsA increased the expression of PIN mRNA. In this study we explored the essential role of PIN in the response of LPS-inducted RAW264.7 cell and the regulation of PIN involved in the anti-inflammation effect of EsA; in addition, we designed the peptide with the consensus motifs of (K/R)XTQT or GIQVD , to invest its regulation to phosphorylation of IkB and production of IL-8 in Jurkat cell stimulated by TNF-a.【Methods】Part one : PIN involving in the anti-inflammation effect of EsA1. The potency of EsA on TNF-a stimulated by LPS in RAW264.7 cells:a) Ascending concentration of EsA affected on RAW264.7 cells pre-stimulated by LPS (1ug/ml)b) Different working-time of EsA affected on RAW264.7 cells pre-stimulated by LPS (1ug/ml)2. The potency of EsA on cAMP levels in RAW264.7 cells.3. The effect of EsA on expression of PIN in RAW264.7 cells in a cAMP-PKA depended-pathway: the protein expression was determined by western blot assay.4. Regulatory role of PIN to inflammation responsesa) Using siRNA targeting PIN and PIN-expressing vector, RAW264.7 cells PIN up- and down- expressing was established. Western blot assay was performed to examine the expression level of PIN.b) TNF-a and NO levels were valued in PIN up-expressed cell or down- expressed cell, respectively.5. PIN involving in the anti-inflammation effect of EsA: expression of TNF-a mRNA was tested by real-time PCR in PIN-interfered RAW264.7 cells. Part two: Effect of the peptides with PIN-binding motif in Jurkat cells1. Ad-peptide and null adenovirus (AdGFP) packaged in 293 cells respectively. The adenoviruses were stored after purification by cesium chloride (CsCl) gradient centrifugation and determination of the viral titers.2. Production of IL-8 was tested in supernant of Jurkat cells transfected with Ad-peptide or AdGFP. ELISA assay was carried out after an additional 4-hour stimulation of TNF-a.3. Phosphorylation level of IkB in Jurkat cells was estimated by western bolting. Jurkat cells were transfected with Ad-peptide or AdGFP followed with an additional 5minues or 10minues'stimulation of TNF-a.【Results】Part one. PIN involving in the anti-inflammation effect of EsA1. Dose-response relationship of EsA to RAW 264.7 cells pre-stimulated with LPS was explored. Concentration of 100uM was selected as a favorable working concentration.2. Time-respones relationship of EsA to RAW 264.7 cells pre-stimulated with LPS was explored as well. 3 hour was the perfect time-window to test the mRNA expression of TNF-a.3. The promotion effect of EsA on cAMP compared with that of forskolin in RAW264.7 cell. There was no difference between them. (p>0.05)4. TNF-a decreased or increased significantly in PIN up-expressed cell or down- expressed cell, respectively; (p<0.05); NO releasing pattern was similar with that of TNF-a in relevant cells.5. The regulatory effect of EsA on TNF-a expression was attenuated significantly in PIN-interfered cells compared with control ones(p<0.05). It was shown that the regulatory effect of EsA to inflammation was PIN-dependant.Part two. Inhibitory effect of the peptide with PIN-binding motif on phosphorylation of IkB and production of IL-8 in Jurkat cells1. The PIN-binding peptide, PIN-binding mutant peptide and control marking protein (GFP) in replication-deficient recombinant adenoviruses were established.2. Administration of Ad-PIN-binding-peptide promoted the production of IL-8 in Jurkat cells significantly, comparing with that of mutant peptide or control. (p<0.05) 3. None of Ad-peptides effected the phosphorylation of IkB.【Conclusion】1. EsA negatively regulated TNF-a in LPS-inducting macrophages. EsA up-regulates the cAMP to promote the transcription of PIN; PIN involved in the down-regulator effect of EsA; the regulatory effect of EsA on inflammation was PIN-dependant.2. PIN-binding peptide promoted the production of IL-8 yet has no effction to phosphorylation of IkB in Jurkat cell.