Research On Treatment Effect Of Pseudolaric Acid B On The Murine Allergic Contact Dermatitis And Its Mechanism

Objective:In this paper, we studied the natural extract of Chinese medicine monomer Pseudolaric acid B (PB) on murine allergic contact dermatitis treatment, and explored its mechanism of action.Methods:1 The cytotoxicity of Pseudolaric acid B to mouse organ and its inhibition effect of proliferation to lymphocyte in vitro by MTT assay.We detected the cytotoxicity of PB to mouse liver, kidney, thymus and Spleen cells in vitro, and the inhibition effect of PB to mouse T lymphocyte proliferation induced by ConA, mouse B lymphocyte proliferation induced by LPS and mixed lymphocyte culture also detected by the method of MTT assay in vitro.2 The effect of Pseudolaric acid B to murine allergic contact dermatitis.By applying different concentrations of pseudolaric acid B(0.1%, 0.2%, 0.4%) , we observed its influence to weight changes, ear swelling, weight difference of ear swelling, ear morphology, thymus index, spleen index, T lymphocyte and B lymphocyte proliferation of murine allergic contact dermatitis.3 The mRNA expression of T-bet, GATA3, ROR-νt, Stat 3,IFN-γ, IL-4, IL-10, TGF-β, IL-6, IL-17, IL-17R in mouse spleen and ear tissues was semi-quantified by reverse transcription PCR (RT-PCR).4 The content of IL-6, IL-10, IL-17 and TGF-βin ear tissues was detected by enzyme linked immunosorbent assay (ELISA).5 The protein expression of Phospho-p-38 MAPK (Thr180/Tyr182), Phospho-Stat 3 (Ser727), Phospho-AKT (ser473) and Phospho-NF-κB p65(ser276) in ear tissues was detected by Western-blot. 6 Observed the role of p-38 MAPK inhibitor to the ear swelling and the difference of ear weight swelling of murine allergic contact dermatitis.Results:1 Activity of Pseudolaric acid B in vitroThe results of MTT assay showed that PB almost had non-toxic to mouse thymus cells in vitro (IC₅₀>1mmol/l), which had an advantage compared with Positive drug hexadecadrol (0.11μmol/L), Cyclosporin (5.23μmol/L), and mycophenolate (23.22μmol/L), and showed hypotoxicity to hepatocyte (64.95μmol/L) and renal cells (73.71μmol/L). PB almost had non-toxic to spleen lymphocyte in 24 and 48 hours in vitro (IC₅₀>1mmol/l), the IC₅₀ in 72 hours was 1.82μmol/L, In contrast, the IC₅₀ of hexadecadrol were 2.59μmol/L, 0.93μmol/l and 0.74μmol/l at the same time. PB inhibited ConA-induced splenic T lymphocyte proliferation and showed a certain time and dose dependence in 24, 48 and 72 hours, these results were similar to hexadecadrol in 48 and 72 hours. PB had a inhibition effect to mixed lymphocyte culture and showed a advantage effect compared with hexadecadrol in 72 hours. PB inhibited LPS-induced splenic B lymphocyte proliferation, however, the results were weaker than that of positive drug. These experiments prove that pseudolaric acid B showed an obvious selective inhibition of T cell proliferation in vitro.2 Treatment of Pseudolaric acid B to murine allergic contact dermatitis.We used different concentrations of pseudolaric acid ointment (0.1%, 0.2%, 0.4%) applied 4 times in the ear of mice, it could be observed that the changes in body weight of mice was increased significantly after 48 hours, medium and high dose group were significantly different than model group (P<0.05). The reductions of ear swelling were significantly different among treated groups and model group after 24 and 48 hours (P<0.05). PB decreased the weight of ear swelling, thymus and spleen index in medium and high dose groups which compared with model group (P<0.05). HE staining of ear 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, however, the medium and high group of PB could reduce the surface damage, tissue edema and infiltration of inflammatory cells. It proved that the medium and high group of PB could significantly inhibit the spleen T lymphocyte proliferation by lymphocyte transformation test (P<0.05), and the high group showed Inhibitory activity to spleen B lymphocyte proliferation (P<0.05). Finally, these results proved that PB showed good treatment to dermatitis and certain selectivity inhibition activity to T cell proliferation in vivo.3 The influence of Pseudolaric acid B to Th1/Th2 balance in allergic contact dermatitis mice.We detected the nuclear factor (T-bet, Gata3) and cytokine (IFN-γ, IL-4) mRNA expression in mice spleen and ear tissues by RT-PCR. The results showed that PB decreased the expression of T-bet and IFN-γ, increased the expression of Gata3 and IL-4 compared with model group after administration, dose dependence was showed in these parts. The regulation of PB to Th1/Th2 balance was proved in these experiments.4 The influence of Pseudolaric acid B to immunoregulatory factor in allergic contact dermatitis mice.We detected the immunoregulatory factor IL-10 and TGF-βmRNA expression in mice spleen and ear tissues by RT-PCR. The results showed that PB increased the expression of IL-10 and TGF-βafter administration. Detected the content of these two cytokine in mice ear tissues, the PB also rised the secretory volume of them and show dose dependence. The immunoloregulation effect of PB to murine allergic contact dermatitis was proved in these experiments.5 The influence of Pseudolaric acid B to inflammation in allergic contact dermatitis mice.We detected the nuclear factor (ROR-νt, Stat 3), cytokine (IL-17, IL-6) and Cytokine receptor IL-17R mRNA expression in mice spleen and ear tissues by RT-PCR. The results showed that PB decreased the expression of them after administration. The PB also reduced the secretory volume of IL-17 and IL-6 in mice ear tissues and show dose dependence according to the result of ELISA. Western blotting experiments showed PB could down regulate 4 signaling pathway proteins (pp-38, p65, pp65, pStat 3) and up regulate pAKT protein which compared with control group to play anti-inflammatory effects. Smear p-38 inhibitor SB203580 to ear of allergic contact dermatitis mice for 4 times in 2 days, we also established positive drug groups of hexadecadrol and Cyclosporin at the same time. We found SB group statistically inhibited the ear swelling statistically significantly compared with control (P<0.05) and with no difference between the two positive drug groups after 24 hours. What's more, the experimental group had significant difference compared with positive drug groups and control group. So we suggested that P38 MAPK pathway prompted an important aspect on this model but not the only aspect. After all, these experiments confirmed pseudolaric acid B affected the relevant signaling pathways to inhibit ACD.Conclusions:Pseudolaric acid B showed good activity of Immunosuppression and Lower toxicity to normal mice cells compared with hexadecadrol Cyclosporin and Mycophenolate in vitro. And it expressed certain selective inhibition of T cells in vivo and in vitro. PB had a good therapeutic effect on the murine allergic contact dermatitis, which may be related with the following channels: it regulated Th1/Th2 balance by decreased T-bet, IFN-γand increased GATA3, IL-4; promoted IL-10 and TGF-βexpression to display immune regulation; inhibited ROR-νt, Stat 3, IL-17R, IL-6 and IL-17 expression to show Anti-inflammatory effects; downregulated Stat 3,MAPK,NF-κB pathway and upregulated AKT pathway to synergy murine allergic contact dermatitis.As a proprietary, new anti-inflammatory and immune regulation of Chinese medicine monomer, Pseudolaric acid B has much better prospects.