| This dissertation was composed of two parts:the literature review and the experimental research.The part of literature review:Literature review one:To form a general understanding about the experimental researches on the treatment of psoriasis with traditional Chinese medicine, the literatures about the experimental researches about the treatment of psoriasis with traditional Chinese herbs and their monomers or compounds in. the recent years were reviewed. They were assorted into six parts, which were the influences from traditional Chinese herbs and their monomers or compounds to the experimental animal model of psoriasis, hemorheology and microcirculation, keratinocyte(KC), fibroblast (FB), cell factor and neuropeptide, to induce and summarize.Literature review two:The literatures about the researches of Rehmannia glutinosa Libosch, Radix Arnebiae and Cortex Moutan and their mainly active ingredients in the recent years were reviewed. And the source, efficacy, chemical compositions that mainly composed of catalpol, shikonin and paeonol, and the overview of pharmacological studies of the three were summarized. Their pharmacological activities are extremely extensive. They also have good development prospects for the treatments of psoriasis.The part of experimental research:.Objective:To study the effective ingredients and the potential mechanisms of action in the treatment of psoriasis with Rehmannia glutinosa Libosch, Radix Arnebiae and Cortex Moutan, the effects to the proliferation of HaCaT cells induced by KGF caused by catalpol,1-shikonin and paeonol, which are the main monomers of the three, are researched.Methods:1. HaCaT cell strain is selected and cultivated in vitro.2. The effects to the proliferation and the cell growth curve of KGF induced HaCaT cells caused by catalpol,1-shikonin and paeonol are detected by CCK-8 assay. 3. The changes of the form of HaCaT cells after drug intervention are observed under a upside down microscope.4. The effects to the cell cycle of KGF induced HaCaT cells proliferation caused by catalpol,1-shikonin and paeonol are detected by a flow cytometry.Results:1. The experimental results of CCK-8 assay showed that after the treatment with KGF, the HaCaT cells proliferation was increased significantly in a dose dependent manner (P<0.05), of which the best concentration is 10ng/ml. The proliferation of HaCaT cells induced by KGF was enhanced by Catalpol at more than or equal to 10-6mol/L concentrat ion (P<0.05). The promotion of prol iferation rate of Catalpol was showed in a dose and time dependent manner, and the best concentration is 10-4mol/L. With the acting time extended, the promotion of proliferation rate of Catalpol was reduced. The proliferation of HaCaT cells induced by KGF was inhibited by 1-shikonin at more than or equal to 10-6mol/L concentration (P<0.05) as well as paeonol at more than or equal to 31.25 mg/L concentration(P<0.05). The inhibition of proliferation rate of 1-shikonin and paeonol was showed in a dose and time dependent manner. As the concentration increased, the inhibiting effect was enhanced. With the acting time extended, the inhibition of proliferation rate of 1-shikonin and paeonol was reduced.2. The cell growth curve showed that HaCaT cells in the treatment with 10ng/ml KGF growed more rapidly compared with control group after the first day of treatment (P<0.01), and after the fifth day of treatment, the growth rate of KGF group showed trend towards control group. The up-regulated growth rate induced by KGF was increased by Catalpol at more than or equal to 10-6mol/L concentration after the first day(P<0.05). And The up-regulated growth rate induced by KGF was depressed by 1-shikonin at more than or equal to 10-6mol/L concentration and paeonol at more than or equal to 31.25 mg/L concentration after the first day(P<0.05).3. The form of HaCaT cells under a upside down microscope showed that the cell density increased and was more intensive in the KGF hole compared with the control hole. The cell density in the hole treated with Catalpol was more intensive than that in the KGF hole. The cell density in the holes treated with 1-shikonin and paeonol were less intensive than that in the KGF hole. There was no significant differences of the HaCaT cells form among each holes.4. The results of the cell cycle showed that after incubated with 10ng/ml KGF for 24 hours, the ratio of S phase and G2M phase of HaCaT cells was increased compared with the control group(P<0.01), and the ratio of G0G1 phase was decreased(P<0.01). The increased S phase and the decreased G0G1 phase induced by KGF was enhanced by Catalpol at more than or equal to 10-6mol/L concentration in a dose dependent manner (P<0.05 and P<0.01), and the best concentration is 10-4mol/L. The ratio of G2M phase had no significant difference campared with KGF group(P>0.05). The increased S phase phase induced by KGF was inhibited by 1-shikonin at more than or equal to 10-6mol/L concentration(P<0.01) as well as paeonol at more than or equal to 31.25 mg/L concentration (P<0.01). The increased G2M phase and the decreased G0G1 phase induced by KGF was inhibited by 1-shikonin at more than or equal to 10-7mol/L concentration (P<0.01) as well as paeonol at more than or equal to 15.625 mg/L concentration(P<0.01). The inhibiting effect of 1-shikonin and paeonol was in a dose dependent manner. As the concentration increased, the inhibiting effect was enhanced. There is no apoptotic peak could be seen in each group.Conclusion:1. The proliferation of HaCaT cells could be induced by KGF by means of increasing the S phase and G2M phase cells. It could simulate in vitro the over proliferative state of epidermal KC in psoriasis. The proliferative model of HaCaT cells induced by KGF is a ideal model for research in vitro about psoriasis.2. The proliferation of HaCaT cells induced by KGF could be inhibited by 1-shikonin and paeonol by means of preventing the transformation of HaCaT cells into S phase and G2M phase, and the growth as well as the proliferation of HaCaT cells would be at a standstill in G0G1 phase, which might be the mechanisms of action in the treatment of psoriasis with Radix Arnebiae and CortexMoutan, and the the effective ingredients might be 1-shikonin and paeonol. The proliferation of HaCaT cells induced by KGF could be enhanced by Catalpol by means of increasing the number of HaCaT cells into S phase, and it could not treat psoriasis by means of inhibiting the proliferation of KC. The effective ingredient and the mechanisms of action in the treatment of psoriasis with Catalpol would be further explored from other angle. |
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