Research On Treatment Effect Of Pseudolaric Acid B On The NC/Nga Mice Atopic Dermatitis And Its Mechanism

Objective:In this paper, we aim to explore the effects and mechanisms of Pseudolaric acid B(PB), a diterpene acid isolated from the root bark of the medicinal plant Pseudolarix amabilis, which has been prescribed in traditional Chinese medicine for centuries, on atopic dermatitis(AD) using NC/Nga mice.Method:1 In vivo1.1 The effects of PB on NC/Nga miceWe observed the influence of PB(5mg/kg, 10mg/kg, 20mg/kg) on the weight changes, dermatitis scores, skin morphology, spleen index, serum Ig E of NC/Nga mice.1.2 The m RNA expression of T-bet, GATA3, IFN-?, IL-4, ASC, caspase-1, IL-1?, NLRP3, ppar-?, ppar-?, TLR-2, TLR-4, IL-17 a, IL-17 Ra, IL-22 m RNA in spleen and skin of AD mice was measured by reverse transcription PCR(RT-PCR).1.3 The levels of IL-1?, IL-17 and IL-22 in serum were detected by enzyme linked immunosorbent assay(ELISA).1.4 The protein expression of Phospho-AKT(Ser473)?RSK-2 and Phospho-?-?B?(Ser32/Ser36) in spleen of AD mice was detected by Western-blot.2 In vitro2.1 The cytotoxicity and inhibition of PB to mouse organ and its inhibition effect on proliferation tolymphocytes were measured by MTT assay in vitro.Firstly, we detected the cytotoxicity of PB to mouse Spleen cells in vitro. Then, the inhibition of PB on the proliferationof T lymphocytes and B lymphocytes induced by Con A and LPS were also detected by MTT assay in vitro.2.2 The activation of PPAR-? in RAW264.7 cells was detected using a reporter gene assay.Results:The results showed that treatment of PB to NC/Nga mice could lead to a marked improvement to AD including the changes of body weight, dermatitis scores and spleen index. H&E staining of skin biopsy revealed that there was significant infiltration of inflammatory cells in interstitial edema and the basal layer and spinous layer of epidermal in model group, and the middle and high doses of PB could reduce the surface damage, tissue edema and infiltration of inflammatory cells. Finally, these results proved that PB showed a good curative effect on AD.The results of RT-PCR and ELISA showed that PB could decrease the expression of IL-1?, TNF-?, NLRP3 and Caspase-1 m RNA compared with the model group dose-dependently. The expression of IL-22 and IL-17 a m RNA in spleen and skin of AD mice was reduced obviously by PB. Moreover, PB could inhibit the m RNA expression of TLR-2 and TLR-4 in AD mice and, block the activation of NF-?B signaling pathway, which would lead to reduce inflammatory cytokine secretion. PB could also promote the expression of PPAR-? and PPAR-? m RNA. These results suggested that PB could play an important role on immunosuppression to reduce inflammation, and alleviate the immune unbalance in AD mice.The results of western blot showed that PB could down regulate the RSK-2, phosphorylation of AKT and ??B? compared with the control group with a dose-dependent manner.The results of PB in vitro showed no cytotoxicity to spleen lymphocytes(IC50>1mmol/L). However, the IC50 of mycophenolate mofetil(MMF) was 22.39?mol/L. Furthermore, PB inhibited the proliferation of splenic T lymphocytes induced by Con A time and dose-dependently, which results were similar to MMF. Though PB could also suppress LPS-induced splenic B lymphocyte proliferation, the inhibition was lower than that of positive drug. These data proved that PB showed an obvious selective inhibition on T cells proliferation in vitro. Finally, the results of luciferase assay showed that PB can increase the PPAR-? activation.Conclusion: PB has the prospect to be a new anti-inflammatory and immunosuppressive agents.