Study On The Inhibition Of Main Components Of Qinbai Ointment On The Proliferation And Activation Of Jurkat T Cell

Psoriasis is a common chronic inflammatory hypertrophic dermatosis. The disease usually has recurrent attacks and is hard to be cured. It is one of the focal points of dermasurgery study. A great progress has been made recently in the study of the nosogenesis of Psoriasis. It is regarded as that Psoriasis is a dysimmunological disease under the polygenic inheritance background, and the abnormal activation and the cytokines play the leading role in this process. The study on the mechanism of T cell activation and its regulation in the psoriasis has been become the hotspot in the research on the psoriasis. Qibai ointment was made up of the radix scutellariae and phellodendron, it has the function of clearing heat, dispelling dampness, reducing swelling, alleviating pain, nourishing blood, invigorating blood, cooling blood and resolving toxins. It is widely used to treat the acute and subacute eczema, psoriasis, impetigo herpetifomis and many dermatoses in clinic. It is an effective medicine for external use to treat psoriasis, which was independently developed by a veteran practioner of TCM, Zhao Bingnan. Now it is the in-house pharmaceutical preparation of Beijing TCM Hospital, it has a great effect to treat the blood heat pattern of psoriasis working in conjunction with the cooling and invigorating blood decoction in clinic.Jurkat T cell was used as a model in this study. The effect of the main components of Qinbai ointment, berberine, scutelloside, scutellarin and wogonin on the activation and proliferation of T cell was investigated. And provide an experimental foundation for the mechanism of treating psoriasis with Qinbai ointment.Experiment 1:The effect of berberine, scutelloside, scutellarin and wogonin on the proliferation of Jurkat T cell. After the cells were treated by drugs for 24h, the cell proliferation was checked by MTT method. The results showed that in vitro, berberine (40mg·L-1,20mg·L-1, 10mg·L-1), scutelloside (200mg·L-1, 100m·L-1L, 50mg·L-1), scutellarin(54mg·L-1,27mg·L-1,13.5mg·L-1) and wogonin (8mg·L-1,4mg·L-1) could inhibit the proliferation of Jurkat T cell after 24h.Experiment 2:Investigate the effect of berberine, scutelloside, scutellarin and wogonin on the secretion of IL-2,IFN-γ,TNF-αfrom Jurkat T cell. The secretion of cytokines was detected by ELISA method after the cells were treated for 8h. The results showed that berberine has an obvious inhibition on the secretion of IL-2,IFN-γand TNF-α, scutelloside has an obvious inhibition on the secretion of IL-2 and IFN-γ, scutellarin has an obvious inhibition on the secretion of IFN-γand TNF-α, wogonin has no inhibition on these three cytokine, but could stimulate the secretion of IL-2.Experiment 3:The effect of berberine, scutelloside, scutellarin and wogonin on the expression of early marker CD69 and middle marker CD25 of T cell activation. The expression of CD69 and CD25 on the Jurkat T cell was detected by single fluorescence staining FACS method after the cells were stimulated by PDB (phorbol 12,13-dibutyrate+Ion (Ionomycin) for 4h and 8h to investigate the effect of berberine, scutelloside, scutellarin and wogonin on the activation of Jurkat T cell in vitro. Thre results showed that in vitro, berberine (40mg·L-1, 20mg·L-1, 10mg·L-1), scutelloside (200mg·L-1, 100mg·L-1, 50mg·L-1), scutellarin (54mg·L-1,27mg·L-1,13.5mg·L-1) and wogonin (8mg·L-1,4mg·L-1) could inhibit the expression of CD69 on the Jurkat T cell when stimulated by PDB+Ion, and showed the dose dependence, but for the CD25, only berberine and wogonin showed the obvious inhibition.Experiment 4:The effect of berberine, scutelloside, scutellarin and wogonin on the activation signal of T cell:dissociated Ca2+ and PKC. The Ca2+ and PKC were detected by single fluorescence staining FACS and ELISA method after the Jurkat T cell was stimulated by PDB+Ion for 24h. The results showed that these four monomeric compound could increase the dissociated Ca2+ and inhibit the expression PKC in cells in various degree.The results showed:①Berberine, scutelloside, scutellarin and wogonin could inhibit the proliferation of Jurkat T cell obviously, and berberine scutelloside and scutellarin could inhibit the secretion of cytokines obviously;②Berberine, scutelloside, scutellarin and wogonin could inhibit the expression of the activation marker CD69 and CD25 of Jurkat T cell obviously;③Berberine, scutelloside, scutellarin and wogonin could increase the expression of T cell activation signal Ca2+, but could effectively inhibit the expression of PKC in T cell. The results suggest that the Qinbai ointment might treat psoriasis through inhibiting the T cell activation PKC signal pathway to inhibit the activation, proliferation and the secretion of cytokines of T cell. This could be one of the mechanisms of the treatment.