| Objective:optimization of optimum dose combination of active components of rhizoma zedoariae and rhizoma sparganii to the inhibition of proliferation of HaCaT cells (immortalized epithelial cells), and the research of its inhibition mechanism, in order to the further study of the drug provide scientific theory basis for the treatment of psoriasis.Methods:(1) Using the method of MTT and the lab-grown HaCaT cells, with different concentrations of IFN-gamma (0,1,5,10,20,50,100 ng mL-1) influencing on HaCaT cell proliferation, choosing the best concentration of promoting HaCaT cell proliferation of IFN-gamma;(2) Using the method of MTT to detect the influence on the proliferation of HaCaT cell about jimma methadone, isoborneol, borneol, curcumin, rhizoma zedoariae diketone, curcumenol, zedoary turmeric oil, beta carotene in the rhizoma zedoariae and formononetin, vanillic acid, vanillin, palmitic acid.kaempferide, succinic acid, linoleic acid and rutin in the rhizoma sparganii by different concentrations (0,5,10,20,50,100 mol/L) and different time (24 h, 48 h,72 h) promoting the proliferation HaCaT cells by the best concentration of IFN-gamma, and set the same concentration of IFN-gamma group and blank group.(3) By the method of MTT and orthogonal experiment to select the best combination of active ingredients of rhizoma zedoariae and rhizoma sparganii about the inhibition rate of the proliferation of HaCaT cell, and set the blank group and the IFN-gamma group, and validate the result of the orthogonal experiment.(4) Using ELISA method to detect IL-2 and TLR-2 levels of HaCaT cell secretion of the group of the best combination of active ingredients of rhizoma zedoariae and rhizoma sparganii, various effective monomer composition (vanillic acid, succinic acid, isoborneol and curcumin), IFN-gamma and blank.(5) Using the method western blot to observe the signal channel protein expression of hedghog PTCH1 of HaCaT cell of the group of the best combination of active ingredients of rhizoma zedoariae and rhizoma sparganii, various effective monomer composition (vanillic acid, succinic acid, isoborneol and curcumin), IFN-gamma and blank.Results:(1) Compared with the blank group, IFN-gamma density dependence on HaCaT cells proliferation, concentration of 5 ng·mL-1 IFN gamma having a significant effect on promoting proliferation on HaCaT cells(P <0.05), the concentration of 50 ng·mL-1 IFN-gamma effecting on promoting proliferation of HaCaT cells is most obvious, and to the basic maximum effect (P<0.01).(2) Compared with the IFN-gamma group, the ingredients of jimma ketone, borneol, rhizoma zedoariae diketone, curcumenol, beta-carotene and formononetin, vanillin, palmitic acid,kaempferide, linoleic acid and rutin in 24h, 48h,72h of different concentration has no obvious inhibition on proliferating HaCaT cell; While all concentration of zedoary turmeric oil in 24h and the concentration of isoborneol, curcumin, succinic acid, vanillic acid in 24h of 10 mol·L-1,20mol·L-1,50mol·L-1, 100mol·L-1 inhibiting the proliferation of HaCaT cell, of which the concentration of zedoary turmeric oil in 24h 20mol·L-1 and 50mol·L-1 and isoborneol in 24h 20mol·L-1 have the strongest inhibitory effect on HaCaT cells. Therefore, according to the L9 (34) orthogonal experiments,choose the active components:rhizoma zedoariae (isoborneol and curcumenol), rhizoma sparganii (vanillic acid and succinic acid) and three dose(10 mol·L-1,20mol·L-1,50mol·L-1) in 24h.(3) The primary and secondary relations of orthogonal experiment is:succinic acid> curcumin> isoborneol> vanillic aci. The best combination of active components of rhizoma zedoariae and rhizoma sparganii is:vanillic acid 50mol·L-1, succinic acid 50mol-L-1, isoborneol 50mol·L-1 and curcumin 50mol·L-1(4) ELISA experiment results:compared with the blank group, IL-2 content of the IFN-gamma group had significant differences (P< 0.01); TLR-2 content of the INF-gamma group difference (P< 0.05); Each drug group of IL-2 content had significant difference (P< 0.001), was significantly reduced, the content of TLR-2 drug group was significantly different (P< 0.01). IL-2 and TLR-2 of group with the best combination of active components of rhizoma zedoariae and rhizoma sparganii was least.the IFN-gamma group had the most abundant IL-2 and TLR-2.(5) Westren blot experiments showed that each drug group effecting on HaCaT cells can cut hedghog signal channel protein PTCH1 expression. The best combination of active components of rhizoma zedoariae and rhizoma sparganii had the weakest PTCH1 expression, and the IFN-gamma group had the strongest expression of protein PTCH1.Conclusion:(1) Isoborneol and curcumin in rhizoma zedoariae and vanillic acid and succinic acid in rhizoma sparganii can inhibit the proliferation of HaCaT cell. The best combination of active components of rhizoma zedoariae and rhizoma sparganii has obvious inhibitory effect on the proliferation of HaCaT cell;(2) IFN-gamma can strengthen the secretion of IL-2 and TLR-2 in HaCaT cells; (3) Each drug group can significantly reduce the secretion of IL-2 and TLR-2 of HaCaT cells and the best combination of active components of rhizoma zedoariae and rhizoma sparganii reduce the most secretion of IL-2 and TLR-2. IL-2 and TLR-2 may play an important role in the abnormal proliferation of active HaCaT cell in psoriasis. Rhizoma zedoariae and rhizoma sparganii for treating psoriasis may be related to the active components of Rhizoma zedoariae and rhizoma sparganii to inhibit HaCaT cells to secrete IL-2 and TLR-2.(4) Each drug group can cut the expression of PTCH1 protein in hedghog signal channel, and the best combination of active components of rhizoma zedoariae and rhizoma sparganii has the strongest action of cutting the expression of PTCH1. This indicate that rhizoma zedoariae and rhizoma sparganii for treating psoriasis may be related to blocking hedghog signal channel and downgrading the expression of PTCH1 protein. |
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