The Effect And Molecular Mechanism Of Paeoniflorin On T Lymphocytes In Allergic Contact Dermatitis Mice

Allergic contact dermatitis（ACD）is a onset hypersensitivity induced by T lymphocytes in the skin and mucosa after exposure to allergens.It is clinically characterized by erythema,papules,blisters and scales,accompanied by severe itching.ACD lesions are widely distributed,usually in a symmetrical distribution,very easy to relapse,and great harm to the physical and mental health of patients.In system drugs for the treatment of ACD,antihistamines are usually only symptomatic treatment,and steroid hormones and immunosuppressants are not suitable for long-term use due to their obvious side effects.Therefore,effective treatments with fewer side effects are urgently needed for ACD.In recent years,a large number of studies have proved that traditional Chinese medicine can effectively improve ACD symptoms through its unique anti-inflammatory and immunomodulatory effects,and no significant side effects have been found^[1].Therefore,effective and low-toxic traditional Chinese medicines have attracted the more attention of researchers.Paeoniflorin（PF）,also known as peonidin,is an active monoterpene glycoside extracted from the root of paeonialactiflora.As an effective anti-inflammatory and immunomodulatory drug,it can participate in the regulation of NF-κB,STAT3 and other inflammation-related signalling pathways,influence immune cells function,and treat various inflammatory diseases,including ACD,rheumatoid arthritis,colitis,etc^[2-5].The previous research confirmed that PF can promote the apoptosis and inhibit the proliferation of activated human peripheral blood T lymphocytes,and iinhibit the expression level of interferon-γ（IFN-γ）in the supernatant of T lymphocytes in a dose-dependent manner.Besides,the nuclear factorκB（NF-κB）and p38mitogen-activated protein kinase（p38 MAPK）signaling pathways may be involved in the PF-mediated immunosuppressive effect on T lymphocytes^[6].In order to further study whether PF attenuates the ACD response by inhibiting the level of IFN-γand the activation of NF-κB and p38 MAPK signaling pathways in T lymphocytes,this study used the DNCB-induced ACD mice model to learn the effect and molecular mechanism of PF on T lymphocytes in ACD mice in vivo.Objective:To study the effect of PF on cytokines and the key pathway proteins in the NF-κB/MAPK signaling pathway,and its anti-inflammatory mechanism in T lymphocytes of ACD mice.Methods:1.The establishment of DNCB-induced ACD mice model and the therapeutic effect of paeoniflorin on ACD mice.Balb/c mice were randomly divided into 5 groups,which were normal control group,model group,low-dose PF group,high-dose PF group,and hydrocortisone（Hyd）group,with 8 animals in each group,and intragastric treatment was performed on days 1to 7.The ear thickness difference,ear swelling weight difference,ear tissue morphology and spleen index of each group were detected during the experiment.2.The effect of PF on cytokines in T lymphocyte supernatant of ACD mice.The contents of IFN-γ,IL-4,IL-17 and IL-31 in the supernatant of T lymphocytes were determined by ELISA.3.The effect of PF on the expression of the key pathway proteins of NF-κB and MAPK signaling pathway in T lymphocytes of ACD mice.The protein levels of NF-κB p65,IκBα,p-IκBα（NF-κB pathway）,and the protein levels of p38,p-p38,JNK,p-JNK,ERK,p-ERK（MAPK pathway）in T lymphocytes of each group were determined by Western Blotting.Results:1.PF inhibited the DNCB-induced ACD response.Compared with the ACD model group,the spleen index in the low-dose and high-dose PF group was significantly reduced（P<0.05,P<0.01）;In a dose-dependent manner,PF significantly reduced the difference of ear thickness and ear swelling weight in ACD mice（P<0.01）,inhibited the proliferation of T lymphocytes in ACD mice（P<0.01）;HE stain of tissue of ear skin injury in mice showed that the ACD model group induced by DNCB showed obvious epidermal hyperkeratosis and inflammatory cell infiltration,while the low and high dose PF group showed significantly reduced epidermal hyperkeratosis and inflammatory cell infiltration.2.PF inhibited the secretion of IFN-γ,IL-17 and IL-31 from T lymphocytes of ACD mice.The levels of inflammatory factors were determined by ELISA.The results showed that the secretion of IFN-γ,IL-17 and IL-31 in T lymphocytes of ACD model group induced by DNCB increased significantly（P<0.01）,and the level of IL-4 had no significant change（P>0.05）.PF significantly decreased the secretion of IFN-γ,IL-17and IL-31 in a dose-dependent manner（P<0.01）,but had no significant effect on IL-4（P>0.05）.3.The effect of PF on the NF-κB/MAPK signaling pathway in T lymphocytes of ACD mice.The protein level of NF-κB p65（P<0.05）and the phosphorylation levels of IκBα,p38,JNK and ERK（P<0.01）were significantly increased in DNCB induced ACD model group.Meanwhile,Compared with model group,the protein level of NF-κB p65and the phosphorylation level of IκBαwere significantly decreased in low and high dose PF group（75 or 150 mg/kg.d）（P<0.01）,but the activation of MAPK signaling pathwaywas not significantly affected（P>0.05）.Conclusion:1.PF inhibits the secretion of IFN-γ,IL-17 and IL-31 by T lymphocytes in ACD mice in a concentration-dependent manner,suggesting that PF may attenuate the ACD immune response by inhibiting the expression of pro-inflammatory factors.2.PF inhibits NF-κB p65 and p-IκB-αprotein levels in T lymphocytes of ACD mice in a concentration-dependent manner,but has no effect on the activation of key proteins in the MAPKs pathway,suggesting that PF may reduce NF-κB/IκB-αconduction pathway suppresses the ACD immune response.3.PF as an effective T lymphocyte targeted drug for the treatment of ACD is worthy of further exploration.