消银解毒饮治疗寻常型银屑病的细胞生物学研究
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摘要
银屑病是一种以红斑、鳞屑为主要表现的慢性炎症性皮肤病,在我国和全世界都是常见病、多发病。当前银屑病的病因和发病机制尚未完全得以阐明,治疗仍然存在疗效不满意和易于复发的问题。现代研究表明,银屑病的基本病理表现为表皮角质形成细胞的过度增生和分化不全,以及真皮毛细血管的增生。我院内制剂——消银解毒饮在我科临床应用二十余年,取得了良好的疗效。我科近期完成国家中医药管理局重点科研课题临床研究显示,其治疗银屑病的总有效率为91.8%,显著优于复方青黛胶囊对照组;用药治疗4周后,患者鳞屑中IL-8含量较治疗前明显下降。在动物实验研究证实,其对SEB诱导的小鼠血清IL-8的升高有拮抗作用,具有显著的免疫调节功能。为探讨消银解毒饮除通过免疫调节而发挥其治疗作用外,是否尚具有对角质形成细胞的直接作用;对真皮血管内皮细胞有何影响;以及消银解毒饮中凉血、解毒、祛风除湿等三组主要成分在治疗银屑病时所起的作用。我们运用药理血清的研究方法,以角质形成细胞株COLO-16和人脐静脉内皮细胞株ECV-304为研究对象,用四唑盐(MTT)比色法观察消银解毒饮及拆方后各组药物血清对其增殖的影响;用流式细胞技术观察药物对其凋亡的影响;用双抗体夹心ABC-ELISA法测定药物对角质形成细胞分泌VEGF的影响。通过细胞生物学方面的研究,进一步探讨了消银解毒饮治疗银屑病的作用机制和作用靶位。并为中医辨证组方规律的研究以及银屑病的临床治疗提供新的思路和方法。
结果显示:①MTX组较蒸馏水组细胞存活率明显降低,随着浓度的升高差异亦加大,在浓度高于10%时二者有极其显著性差异(P<0.001)。消银解毒饮组和凉血方组体外培养角质形成细胞COLO-16的存活率明显降低,且细胞的存活率与含药血清浓度呈负相关,浓度越高存活率越低。消银解毒饮组在浓度为5%、10%时较蒸馏水组有显著性差异(P<0.05),当浓度升高到20%时有极其显著性差异(P<0.001)。凉血方组在浓度为10%时较蒸馏水组有显著性差异(P<0.05),当浓度升高到20%时有极其显著性差异(P<0.001)。而解毒方组和祛风除湿方组随着含药血清浓度的升高体外培养角质形成细胞COLO-16的存活率有所降低,但各个浓度组与蒸馏水相比无显著性差异(P>0.05)。②在含药血清浓度为20%时,解毒方组、祛风除湿方组、凉血方组等三组细胞的凋亡率与蒸馏水组比较无显著性差别(P<0.05);而消银解毒饮组、MTX组凋亡率显著增高,与蒸馏水组相比有极为显著性差异(P<0.001)。③在5%浓度下,MTX组、消银解毒饮组和凉血方组体外培养血管内皮细胞的存活率均有所降低,但只有MTX组与对照组比较有较为显著的差别(P<0.05)。在10%浓度下,消银解毒饮组、凉血方组及MTX组血管内皮细胞的存活率均有所下降,其中消银解毒饮组和MTX组细胞存活率下降的较为明显,与对照组比较有极其显著性差异(P<0.001),凉血方组与对照组比较亦有较为显著的差别(P<0.05)。在20%浓度下,消银解毒饮组、祛风除湿方组、凉血方组以及MTX组血管内皮细胞的存活率均有下降,其中消银解毒饮组、凉血方组及MTX组与对照组比较有极其显著性差异(P<0.001)。祛风除湿方组有较为显著差异(P<0.05)。④在5%浓度下,MTX组和消银解毒饮组对体外培养角质形成细胞COLO-16分泌VEGF有抑制作用,消银解毒饮组的抑制作用更为明显,与对照组相比有极其显著的差异。在10%浓度下,消银解毒饮组、解毒方组及MTX组对COLO-16细胞分泌VEGF均有抑制作用,其中消银解毒饮组和解毒方组与对照组比较有极其显著性差异(消银解毒饮组P<0.001,解毒方组P <0.01)。在20%浓度下,消银解毒饮组、解毒方组、凉血方组以及MTX组对COLO-16细胞分泌VEGF均有抑制作用,其中消银解毒饮组、解毒方组及MTX组与对照组比较有极其显著性差异(P<0.001)。消银解毒饮组、解毒方组凉血方组及MTX组随着含药血清浓度的升高VEGF含量降低,两者呈负相关。
结果表明:①消银解毒饮能抑制银屑病患者角质形成细胞的异常增殖并诱导其凋亡。②消银解毒饮对患者血管内皮细胞的过度增生亦具有的抑制作用。③消银解毒饮还可抑制患者角质形成细胞分泌血管内皮细胞生长因子。④在消银解毒饮的拆方中,凉血药物对抑制细胞的过度增殖发挥主要作用,解毒药物对抑制角质形成细胞分泌VEGF起主要作用;而三组药物的协同配合可使各种作用明显加强,从而对银屑病的有效治疗发挥更好的作用。⑤消银解毒饮除通过免疫调节而发挥其治疗作用外,还可通过对角质形成细胞过度增殖的直接抑制及诱导其凋亡,抑制真皮微血管的异常增生而发挥治疗作用。消银解毒饮可通过多个环节,多个靶点对银屑病发挥治疗作用。为临床运用消银解毒饮治疗银屑病提供了重要的理论依据。
Psoriasis(PS) is a chronic inflammatory dermatosis characterized by erythema and scales. As a common disease throughout the world it affects patients’physical and mental health greatly. The aetiology of PS is not clear yet and the curative effect is not very satisfatory. Usually, it has a protract course, liable to relapse. Modern researches have established hyperkeratinization and parakeratosis of keratinocytes(KC) and the hyperplasia of the capillary vessel in the dermis as the fundamental pathological changes.
Xiao Yin Jie Du Decocction (XYJDY) is an effective herbal medicine manufactured by our hospital and it has been used to treat PS for more than twenty years in our department. Clinic studies finished by our department recently has shown that the total effective rate is 91.8%, more than that of the control group using Fu Fang Qing Dai Capsule. Interleukin-8(IL-8) in the scales of psoriasis patients decreased significantly after four weeks’treatment of XYJDY. Animal experiment indicated that XYJDY could also inhibit IL-8 production and antagonize IL-8 increase induced by SEB in mouse’s blood. In order to study the effect of XYJDY on KC and the endothelium of hypoderma and observe the curative effect of three main components group of XYJDY (cooling blood Group、detoxicating Group、expelling wind and removing dampness Group), Using MTT chromatometry, we studied the effect of mouse serum (treated with XYJDY and modified decoction) on the multiplication of Malpighian cell COLO-16 and human navel intravenous endotheliocyte ECV-304. We studied their apoptosis with flow cytometry and VEGF secretion of malpighian cell with double antibody ABC-ELISA method. We probed into the mechanism and the target site of XYJDY in treating PS by using the cytobiological research methods in order to explore new methods for the clinic research of PS and theorectical research of syndrome differentiation.
The findings suggested that: 1.XYJDY and cooling blood group could significantly restrain the proliferation of KC The higher concentration of decoction was , the lower the survival rate would be . The survival rate of KC in XYJDY group (concentration of 5% and 10% ) was significantly lower than that in the control group of distilled water (P<0.05), and even lower when the concentration of XYJDY is 20%(P<0.001). The survival rate of KC in cooling blood group was significantly lower than that in the control group when the concentration of the herb was 10%(P<0.05), and even lower when the concentration was 20% (P<0.001). The survival rate of KC in detoxicating group or expelling wind and removing dampness group was not statistically different from that in the control group, but the higher concentration of decoction was , the lower survival rate became. There was significant difference (P<0.001) in the survival rate of KC between MTX group and the control group when the concentration of MTX was higher than 10%. 2. The apoptosis of KC in detoxicating group, expelling wind and removing dampness group and cooling blood group was not significantly different from that in the control when the concentration was 20%. The apoptosis of KC in XYJDY group or MTX group was significantly higher than that in the control group (distilled water) (P<0.001) 3. With a serum medicine concentration of 5%, the sruvival rate of endothelial cell in MTX group、XYJDY group and cooling blood group were all lower than that in the control group, but sruvival rate of endothelial cell in MTX group only was significantly lower than that in the control group(P<0.05). When the concentration of medicine reached 10%, the sruvival rate of endothelial cell in MTX group and XYJDY group were extremely significantly lower than that in the control group(P<0.001), and in cooling blood Fang group it was significantly lower than that in the control group(P<0.05). When the concentration of medicine reached 20%, the sruvival rate of endothelial cell in MTX group、XYJDY group and cooling blood Fang group were all extremely significantly lower than that in the control group(P<0.001), and in expelling wind and removing dampness group it was significantly lower than that in the control group(P<0.05). 4. When the concentration of medicine was 5%, XYJDY and MTX group could restrain the VEGF released by colo-16 cell in vitro, and the level of VEGF in XYJDY group was significantly lower than that in the control group. When the concentration of medicine was 10%, XYJDY group、MTX group and detoxicating group could restrain the VEGF released by colo-16 cell in vitro, and the level of VEGF in XYJDY group and detoxicating group was significantly lower than that in the control group. When the concentration of medicine was 20%, XYJDY group、cooling blood group、MTX group and detoxicating group could all restrain the VEGF released by colo-16 cell in vitro, and the level of VEGF in XYJDY group、cooling blood group and detoxicating group was significantly lower than that in the control group. The findings shows that VEGF level has an inverse association with serum concentration of medicine in XYJDY group、cooling blood group、MTX group and detoxicating group.
The result indicates that:1. XYJDY can restrain the hyperplasia of keratinocyte and induce apoptosis of keratinocyte in psoriasis patients. 2. XYJDY can also restrain the hyperplasia of vascular endothelial cell of psoriasis patients. 3. XYJDY can inhibit the production of VEGF, released by keratinocyte. 4. By separating XYJDY to three main component groups to observe the effect of them on psoriasis. We find that cooling blood group can restrain the hyperplasia of keratinocyte and blood vessel endothelium cell while detoxicating group can inhibit the production of VEGF released by keratinocyte, and that the combination of three groups could enhance these effects significantly. 5. The findings indicated that the curative effect of XYJDY on psoriasis is probably mediated by inhibiting KC proliferation、inducing apoptosis of keratinocyte and restraining the hyperplasia of dermis capillary vessel. We postulate that XYJDY’s therapeutic effect on psoriasis is probably realized through different target sites.
结果显示:①MTX组较蒸馏水组细胞存活率明显降低,随着浓度的升高差异亦加大,在浓度高于10%时二者有极其显著性差异(P<0.001)。消银解毒饮组和凉血方组体外培养角质形成细胞COLO-16的存活率明显降低,且细胞的存活率与含药血清浓度呈负相关,浓度越高存活率越低。消银解毒饮组在浓度为5%、10%时较蒸馏水组有显著性差异(P<0.05),当浓度升高到20%时有极其显著性差异(P<0.001)。凉血方组在浓度为10%时较蒸馏水组有显著性差异(P<0.05),当浓度升高到20%时有极其显著性差异(P<0.001)。而解毒方组和祛风除湿方组随着含药血清浓度的升高体外培养角质形成细胞COLO-16的存活率有所降低,但各个浓度组与蒸馏水相比无显著性差异(P>0.05)。②在含药血清浓度为20%时,解毒方组、祛风除湿方组、凉血方组等三组细胞的凋亡率与蒸馏水组比较无显著性差别(P<0.05);而消银解毒饮组、MTX组凋亡率显著增高,与蒸馏水组相比有极为显著性差异(P<0.001)。③在5%浓度下,MTX组、消银解毒饮组和凉血方组体外培养血管内皮细胞的存活率均有所降低,但只有MTX组与对照组比较有较为显著的差别(P<0.05)。在10%浓度下,消银解毒饮组、凉血方组及MTX组血管内皮细胞的存活率均有所下降,其中消银解毒饮组和MTX组细胞存活率下降的较为明显,与对照组比较有极其显著性差异(P<0.001),凉血方组与对照组比较亦有较为显著的差别(P<0.05)。在20%浓度下,消银解毒饮组、祛风除湿方组、凉血方组以及MTX组血管内皮细胞的存活率均有下降,其中消银解毒饮组、凉血方组及MTX组与对照组比较有极其显著性差异(P<0.001)。祛风除湿方组有较为显著差异(P<0.05)。④在5%浓度下,MTX组和消银解毒饮组对体外培养角质形成细胞COLO-16分泌VEGF有抑制作用,消银解毒饮组的抑制作用更为明显,与对照组相比有极其显著的差异。在10%浓度下,消银解毒饮组、解毒方组及MTX组对COLO-16细胞分泌VEGF均有抑制作用,其中消银解毒饮组和解毒方组与对照组比较有极其显著性差异(消银解毒饮组P<0.001,解毒方组P <0.01)。在20%浓度下,消银解毒饮组、解毒方组、凉血方组以及MTX组对COLO-16细胞分泌VEGF均有抑制作用,其中消银解毒饮组、解毒方组及MTX组与对照组比较有极其显著性差异(P<0.001)。消银解毒饮组、解毒方组凉血方组及MTX组随着含药血清浓度的升高VEGF含量降低,两者呈负相关。
结果表明:①消银解毒饮能抑制银屑病患者角质形成细胞的异常增殖并诱导其凋亡。②消银解毒饮对患者血管内皮细胞的过度增生亦具有的抑制作用。③消银解毒饮还可抑制患者角质形成细胞分泌血管内皮细胞生长因子。④在消银解毒饮的拆方中,凉血药物对抑制细胞的过度增殖发挥主要作用,解毒药物对抑制角质形成细胞分泌VEGF起主要作用;而三组药物的协同配合可使各种作用明显加强,从而对银屑病的有效治疗发挥更好的作用。⑤消银解毒饮除通过免疫调节而发挥其治疗作用外,还可通过对角质形成细胞过度增殖的直接抑制及诱导其凋亡,抑制真皮微血管的异常增生而发挥治疗作用。消银解毒饮可通过多个环节,多个靶点对银屑病发挥治疗作用。为临床运用消银解毒饮治疗银屑病提供了重要的理论依据。
Psoriasis(PS) is a chronic inflammatory dermatosis characterized by erythema and scales. As a common disease throughout the world it affects patients’physical and mental health greatly. The aetiology of PS is not clear yet and the curative effect is not very satisfatory. Usually, it has a protract course, liable to relapse. Modern researches have established hyperkeratinization and parakeratosis of keratinocytes(KC) and the hyperplasia of the capillary vessel in the dermis as the fundamental pathological changes.
Xiao Yin Jie Du Decocction (XYJDY) is an effective herbal medicine manufactured by our hospital and it has been used to treat PS for more than twenty years in our department. Clinic studies finished by our department recently has shown that the total effective rate is 91.8%, more than that of the control group using Fu Fang Qing Dai Capsule. Interleukin-8(IL-8) in the scales of psoriasis patients decreased significantly after four weeks’treatment of XYJDY. Animal experiment indicated that XYJDY could also inhibit IL-8 production and antagonize IL-8 increase induced by SEB in mouse’s blood. In order to study the effect of XYJDY on KC and the endothelium of hypoderma and observe the curative effect of three main components group of XYJDY (cooling blood Group、detoxicating Group、expelling wind and removing dampness Group), Using MTT chromatometry, we studied the effect of mouse serum (treated with XYJDY and modified decoction) on the multiplication of Malpighian cell COLO-16 and human navel intravenous endotheliocyte ECV-304. We studied their apoptosis with flow cytometry and VEGF secretion of malpighian cell with double antibody ABC-ELISA method. We probed into the mechanism and the target site of XYJDY in treating PS by using the cytobiological research methods in order to explore new methods for the clinic research of PS and theorectical research of syndrome differentiation.
The findings suggested that: 1.XYJDY and cooling blood group could significantly restrain the proliferation of KC The higher concentration of decoction was , the lower the survival rate would be . The survival rate of KC in XYJDY group (concentration of 5% and 10% ) was significantly lower than that in the control group of distilled water (P<0.05), and even lower when the concentration of XYJDY is 20%(P<0.001). The survival rate of KC in cooling blood group was significantly lower than that in the control group when the concentration of the herb was 10%(P<0.05), and even lower when the concentration was 20% (P<0.001). The survival rate of KC in detoxicating group or expelling wind and removing dampness group was not statistically different from that in the control group, but the higher concentration of decoction was , the lower survival rate became. There was significant difference (P<0.001) in the survival rate of KC between MTX group and the control group when the concentration of MTX was higher than 10%. 2. The apoptosis of KC in detoxicating group, expelling wind and removing dampness group and cooling blood group was not significantly different from that in the control when the concentration was 20%. The apoptosis of KC in XYJDY group or MTX group was significantly higher than that in the control group (distilled water) (P<0.001) 3. With a serum medicine concentration of 5%, the sruvival rate of endothelial cell in MTX group、XYJDY group and cooling blood group were all lower than that in the control group, but sruvival rate of endothelial cell in MTX group only was significantly lower than that in the control group(P<0.05). When the concentration of medicine reached 10%, the sruvival rate of endothelial cell in MTX group and XYJDY group were extremely significantly lower than that in the control group(P<0.001), and in cooling blood Fang group it was significantly lower than that in the control group(P<0.05). When the concentration of medicine reached 20%, the sruvival rate of endothelial cell in MTX group、XYJDY group and cooling blood Fang group were all extremely significantly lower than that in the control group(P<0.001), and in expelling wind and removing dampness group it was significantly lower than that in the control group(P<0.05). 4. When the concentration of medicine was 5%, XYJDY and MTX group could restrain the VEGF released by colo-16 cell in vitro, and the level of VEGF in XYJDY group was significantly lower than that in the control group. When the concentration of medicine was 10%, XYJDY group、MTX group and detoxicating group could restrain the VEGF released by colo-16 cell in vitro, and the level of VEGF in XYJDY group and detoxicating group was significantly lower than that in the control group. When the concentration of medicine was 20%, XYJDY group、cooling blood group、MTX group and detoxicating group could all restrain the VEGF released by colo-16 cell in vitro, and the level of VEGF in XYJDY group、cooling blood group and detoxicating group was significantly lower than that in the control group. The findings shows that VEGF level has an inverse association with serum concentration of medicine in XYJDY group、cooling blood group、MTX group and detoxicating group.
The result indicates that:1. XYJDY can restrain the hyperplasia of keratinocyte and induce apoptosis of keratinocyte in psoriasis patients. 2. XYJDY can also restrain the hyperplasia of vascular endothelial cell of psoriasis patients. 3. XYJDY can inhibit the production of VEGF, released by keratinocyte. 4. By separating XYJDY to three main component groups to observe the effect of them on psoriasis. We find that cooling blood group can restrain the hyperplasia of keratinocyte and blood vessel endothelium cell while detoxicating group can inhibit the production of VEGF released by keratinocyte, and that the combination of three groups could enhance these effects significantly. 5. The findings indicated that the curative effect of XYJDY on psoriasis is probably mediated by inhibiting KC proliferation、inducing apoptosis of keratinocyte and restraining the hyperplasia of dermis capillary vessel. We postulate that XYJDY’s therapeutic effect on psoriasis is probably realized through different target sites.
引文
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