| Background: Psoriasis is a common, chronic, inflammatory and benign hyperproliferation disease. It is stubborn ,recurrent and refractoriness desease. So far there are no adeal therapy for psoriasis. Recently, retinoids has become one of the effective medicine for treatment of psoriasis. But some severe toxic and side effects limit its clinical use. Some traditional herb Chinese medicine has been used on treatment of psoriasis for a long time in our country following the therapeutic principles of cooling blood, activating blood circulation , expelling wind-evil and regulating diuresis,though their treatment mechanism has not been known clearly till now. Cooling blood and activating blood circulation (CBABC) formula which integrated traditional Chinese medical theories and experimental formulas was developed according to the principles of therapy. The therapeutic effects of the formula on psoriasis have been tested by animal experiments and clinical studies. Huangqi injection includes the major effective components of Huangqi herb. Modern pharmacological studies have indicated its immunoenhencement and immunoregulatory effects. Previous observations have also shown its clinical effects on psoriasis.but the mechanism has not been known clearly.Objective: In this study, the therapeutic effects of retinoids with the CBABC formula on psoriasis were compared. The therapeutic effects of Huangqi injection were also observed when combined with retinoids with the CBABC formula. And the effects of acitretin and Huangqi injection on proliferation ,apoptosis and VDR expression in keratinocyte HaCaT were investigated. The major purpose of the study is to explore the pathogenic mechanism of psoriasis and compared the therapeutic mechanism of acitretin in antipsoriatic and Huangqi injection. The results might provide experimental evidence for the combination of retinoids and traditional herb Chinese medicine.Methods: Clinical study: Two hundred and seventy two psoriatic patients who were diagnosed clinically or by pathologic method were divided into three groups according to the admission time. The 41 cases of the first group were treated with Arotinoid ethylester capsules. The 66 cases of the second group were treated with acitretin capsules. The last 165 patients took CBABC formula. At the same time, some of the patients in every group were treated with Huang-qi injection. Therapeutic effects were determined with PASI scores and comparing the effects. The deference of effective rate between the patients treated with Huang-qi injection and the patients without the injection was statistically analyzed.Experimental study: The expression of VDR was observed in psoriatic patients epidermis by immunohistochemical method and compared wth normal skin. Positive cells were counted under microscope and the positive react degree was half quantitatively determined. Cultured HaCaT cells were exposed to acitretin and Huangqi injection at different concentrations for 24h,48h and 72h. Their effects on cell proliferation were measured by MTT assay. Inhibitory rate and IC50 were calculated. According to the results of MTT assay, HaCaT cells were treated with acitretin and Huangqi injection in proper dose and time. In order to observe the effects of both drugs on Cell cycle and apoptosis, all samples were analyzed by flow cytometry. And morphological changes were observed under transmission electron microscope. Expression of VDR protein was half quantitatively analyzed by Western Blot and compared with negative controlled sample, and expression of VDR mRNA was analyzed by Real-Time RT-PCR .Results: The effective rate of the three groups were 82.9%, 78.8% and 64.2% respectively. And the patients with Huang-qi injection got better therapeutic effects. The rate of efficiency of the group treated with CBABC formula was significantly less the other two groups. But there was not obvious difference between Arotinoid ethylester capsules and acitretin capsules.Both acitretin and Huang-qi injection exerted notable inhibitory effect on proliferation of HaCaT cells. After treated by acitretin for 24, 48, 72 hours, the IC50 was 47.80μg/ml, 27.40μg/ml, 18.46μg/ml respectively. After treated by Huang-qi injection for 24,48,72 hours, the IC50 was 341.0 mg /ml, 252.2 mg /ml, 194.3mg /ml respectively. The inhibition revealed time and dose dependence in effective concentration range.The results of flow cytometry analysis showed that both drugs could arrest HaCaT cell in G1 phase and induce apoptosis. The percentage of cells in G1 phage of samples treated with both drugs were more than negative controlled sample, and that of cells in S/G2 phage were less. Acitretin induced HaCaT cells distinct apoptosis at the concentration of 30μg/ml after 48h. Observation of the treated cells under transmission electron microscope showed characteristic opoptotic morphological changes. The effects of Huangqi injection were that between the concentration of 15μg/ml and 30μg/ml of Acitretin.Immunohistochemical method: Positive keratinocytes were found under microscope both in normal skin and psoriatic lesion. And most of those positive cells lied in basic layer of epidermis especially in psoriatic lesion. It showed that both normal skin and psoriatic lesion express VDR protein. Furthermore, expression of VDR protein in psoriatic lesion was more than normal epidermis(P<0.01).Western blot: Every sample produced the bonds whichβ-actin and VDR reacted respectively to their monoclonal antibodies. The molecular weight was about 42kDa or 58kDa. The result showed that expression of VDR protein in HaCaT cells was decreased after treated by acitretin., but the level of VDR protein in HaCaT cells treated by Huangqi injection was near to negative control HaCaT cells.Real-Time RT-PCR: Relative amount of VDR mRNA expression in every sample of HaCaT cells was analyzed through Real-Time RT-PCR. As a result, the level of VDR protein in HaCaT cells treated by Huangqi injection or acitretin closed to negative control HaCaT cells. It suggested that either Huangqi or acitretin can not change the expression of VDR mRNA in HaCaT cells.Conclution: All of Arotinoid ethylester capsule,acitretin capsule and CBABC formula are effective on treatment of psoriasis. Arotinoid ethylester and acitretin are especially effective compared with that CBABC formula. No obvious deference in curative was found between Arotinoid ethylester and acitretin. Huangqi can increase efficiency on psoriasis while combined with the drugs above.Both acitretin and Huangqi injection can inhibit HaCaT cells proliferation in time and dose dependent manner. They can interfere with HaCaT cell cycle and arrest cells in G1 phage, so that the cells can not get in G/M phage and proliferate. Also acitretin and Huangqi can induce HaCaT cell apoptosis,suggesting that their antipsoriatic mechanism may be relevant to these effects.Both normal skin and psoriatic lesion express VDR protein, and the expression in psoriatic lesion was more than normal epidermis.It was demonstrated that this abnormal expression of VDR protein may be a factor of psoriasis pathogenic mechanism. It may explain the therapeutic effect of 1,25(OH)2D3 on psoriasis at molecular level.Acitretin can decrease expression of VDR protein of HaCaT cells but not VDR mRNA. The results suggested Acitretin promoted degradation of VDR protein or Acitretin activates the RXR and forms heterodimers with VDR so that expression of VDR protein was decreased. It could partly explain the mechanism of Acitretin on psoriasis and be responsible for teratogenicity. Huangqi can not change the expression of VDR in HaCaT cells, so its antipsoriatic mechanism has nothing to do with VDR. The results also provides experimental basis for the combining of Huangqi and acitretin.
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