Abstract：
BACKGROUND: Psoriasis is an immune-mediated chronic inflammatory skin disease. Current research suggests that the long-term persistence and recurrence of psoriasis are closely related to the feedback loop formed between keratinocytes and immune cells, especially in Th 17 or DC cells expressing CCR6. CCL20 is the ligand of CCR6. Therefore, drugs that block the expression of CCL20 or CCR6 may have a certain therapeutic effect on psoriasis. Glycyrrhetinic acid (GA) is the main active ingredient of the plant drug licorice and is often used to treat autoimmune diseases, including psoriasis. However, its mechanism of action is still unclear.
METHODS: Psoriasis like skin lesion model was established by continuously applying imiquimod on the back skin of normal mice and CCR6-/- mice for 7 days. The therapeutic and preventive effects of glycyrrhetinic acid (GA) on the model were observed and compared. The severity of skin injury is estimated through clinical PASI scores and histopathological examination. qRT-PCR and multiple cytoline assay were explored to detect the expression levels of cytokines in animal dorsal skin lesions and keratinocyte line HaCaT cells, respectively. The dermis and epidermis of the mouse back were separated for the detection of CCL20 expression. Transcription factor assay was applied to screen, and luciferase activity assay to validate transcription factors regulated by GA. Technology of surface plasmon laser resonance with LC-MS (SPR-MS), molecular docking, and enzyme activity assay were used to identified the target proteins for GA. Finally, we synthesized different derivatives of 18beta-GA and compared their effects, as well as glycyrrhetinic acid (GL), on the skin lesion of imiquimod-induced mice to evaluate the active groups of 18beta-GA.
RESULTS: 18β-glycyrrhetinic acid (GA) improved IMQ-induced psoriatic lesions, and could specifically reduce the chemokine CCL20 level of the epidermis in lesion area, especially in therapeutic administration manner. The process was mainly regulated by transcription factor ATF2 in the keratinocytes. In addition, GUSB was identified as the primary target of 18βGA. Our findings indicated that the subject on molecular target research of glycyrrhizin should be glycyrrhetinic acid (GA) instead of glycyrrhizic acid (GL), because GL showed little activity in vitro or in vivo. Apart from that, α, β, -unsaturated carbonyl in C11/12 positions was crucial or unchangeable to its activity of 18βGA, while proper modification of C3 or C30 position of 18βGA may vastly increase its activity.
CONCLUSIONS: Our research indicates that 18βGA exerted its anti-psoriasis effect mainly by suppressing ATF2 and downstream molecule CCL20 predominately through α, β, -unsaturated carbonyl at C11/12 position binding to GUSB in the keratinocytes, and then broke the feedback loop between keratinocytes and CCR6-expressing immune cells. GA has more advantages than GL in the external treatment of psoriasis. A highlight of this study is to investigate the influence of special active groups on the pharmacological action of a natural product, inspired by the molecular docking result.
METHODS: Psoriasis like skin lesion model was established by continuously applying imiquimod on the back skin of normal mice and CCR6-/- mice for 7 days. The therapeutic and preventive effects of glycyrrhetinic acid (GA) on the model were observed and compared. The severity of skin injury is estimated through clinical PASI scores and histopathological examination. qRT-PCR and multiple cytoline assay were explored to detect the expression levels of cytokines in animal dorsal skin lesions and keratinocyte line HaCaT cells, respectively. The dermis and epidermis of the mouse back were separated for the detection of CCL20 expression. Transcription factor assay was applied to screen, and luciferase activity assay to validate transcription factors regulated by GA. Technology of surface plasmon laser resonance with LC-MS (SPR-MS), molecular docking, and enzyme activity assay were used to identified the target proteins for GA. Finally, we synthesized different derivatives of 18beta-GA and compared their effects, as well as glycyrrhetinic acid (GL), on the skin lesion of imiquimod-induced mice to evaluate the active groups of 18beta-GA.
RESULTS: 18β-glycyrrhetinic acid (GA) improved IMQ-induced psoriatic lesions, and could specifically reduce the chemokine CCL20 level of the epidermis in lesion area, especially in therapeutic administration manner. The process was mainly regulated by transcription factor ATF2 in the keratinocytes. In addition, GUSB was identified as the primary target of 18βGA. Our findings indicated that the subject on molecular target research of glycyrrhizin should be glycyrrhetinic acid (GA) instead of glycyrrhizic acid (GL), because GL showed little activity in vitro or in vivo. Apart from that, α, β, -unsaturated carbonyl in C11/12 positions was crucial or unchangeable to its activity of 18βGA, while proper modification of C3 or C30 position of 18βGA may vastly increase its activity.
CONCLUSIONS: Our research indicates that 18βGA exerted its anti-psoriasis effect mainly by suppressing ATF2 and downstream molecule CCL20 predominately through α, β, -unsaturated carbonyl at C11/12 position binding to GUSB in the keratinocytes, and then broke the feedback loop between keratinocytes and CCR6-expressing immune cells. GA has more advantages than GL in the external treatment of psoriasis. A highlight of this study is to investigate the influence of special active groups on the pharmacological action of a natural product, inspired by the molecular docking result.
摘要：
背景：银屑病是一种免疫介导的慢性炎症性皮肤病。目前的研究表明，银屑病的长期持续和复发与角质形成细胞和免疫细胞之间形成的反馈回路密切相关，特别是在Th17或表达CCR6的DC细胞中。CCL20是CCR6的配体。因此,抑制CCL20或CCR6表达的药物可能对银屑病有一定的治疗作用。甘草次酸(GA)是植物药物甘草的主要活性成分,常用于治疗自身免疫性疾病,包括牛皮癣。然而,其作用机制尚不清楚。
方法：在正常小鼠和CCR6-/-小鼠背部皮肤上连续应用咪喹莫特7天，建立银屑病样皮肤病变模型。观察并比较甘草次酸（GA）对模型的治疗和预防作用。通过临床PASI评分和组织病理学检查估计皮肤损伤的严重程度。qRT-PCR和多重cytoline法检测细胞因子在动物背部皮肤病变和角质形成细胞系HaCaT细胞中的表达水平,分别。分离小鼠背部的真皮和表皮用于检测CCL20表达。转录因子测定法用于筛选，和荧光素酶活性测定来验证GA调控的转录因子。LC-MS（SPR-MS）表面等离子体激元激光共振技术，分子对接,和酶活性测定用于鉴定GA的靶蛋白。最后,我们合成了18β-GA的不同衍生物，并比较了它们的作用，以及甘草次酸（GL），对咪喹莫特诱导小鼠皮肤损伤的18β-GA活性组进行评价。
结果：18β-甘草次酸（GA）改善了IMQ诱导的银屑病病变，并能特异性降低病变区表皮趋化因子CCL20水平，特别是在治疗给药方式上。该过程主要受角质形成细胞中转录因子ATF2的调控。此外,GUSB被确定为18βGA的主要靶标。我们的发现表明，甘草酸的分子靶标研究的主题应该是甘草次酸（GA）而不是甘草酸（GL），因为GL在体外或体内几乎没有活性。除此之外,α,β,C11/12位的-不饱和羰基对其18βGA的活性至关重要或不变，而适当修饰18βGA的C3或C30位置可能会大大提高其活性。
结论：我们的研究表明，18βGA主要通过抑制ATF2和下游分子CCL20主要通过α，β,-C11/12位置的不饱和羰基与角质形成细胞中的GUSB结合，然后打破角质形成细胞和表达CCR6的免疫细胞之间的反馈回路。GA在银屑病的外用医治方面比GL更具优势。这项研究的重点是研究特殊活性基团对天然产物药理作用的影响，受到分子对接结果的启发。
方法：在正常小鼠和CCR6-/-小鼠背部皮肤上连续应用咪喹莫特7天，建立银屑病样皮肤病变模型。观察并比较甘草次酸（GA）对模型的治疗和预防作用。通过临床PASI评分和组织病理学检查估计皮肤损伤的严重程度。qRT-PCR和多重cytoline法检测细胞因子在动物背部皮肤病变和角质形成细胞系HaCaT细胞中的表达水平,分别。分离小鼠背部的真皮和表皮用于检测CCL20表达。转录因子测定法用于筛选，和荧光素酶活性测定来验证GA调控的转录因子。LC-MS（SPR-MS）表面等离子体激元激光共振技术，分子对接,和酶活性测定用于鉴定GA的靶蛋白。最后,我们合成了18β-GA的不同衍生物，并比较了它们的作用，以及甘草次酸（GL），对咪喹莫特诱导小鼠皮肤损伤的18β-GA活性组进行评价。
结果：18β-甘草次酸（GA）改善了IMQ诱导的银屑病病变，并能特异性降低病变区表皮趋化因子CCL20水平，特别是在治疗给药方式上。该过程主要受角质形成细胞中转录因子ATF2的调控。此外,GUSB被确定为18βGA的主要靶标。我们的发现表明，甘草酸的分子靶标研究的主题应该是甘草次酸（GA）而不是甘草酸（GL），因为GL在体外或体内几乎没有活性。除此之外,α,β,C11/12位的-不饱和羰基对其18βGA的活性至关重要或不变，而适当修饰18βGA的C3或C30位置可能会大大提高其活性。
结论：我们的研究表明，18βGA主要通过抑制ATF2和下游分子CCL20主要通过α，β,-C11/12位置的不饱和羰基与角质形成细胞中的GUSB结合，然后打破角质形成细胞和表达CCR6的免疫细胞之间的反馈回路。GA在银屑病的外用医治方面比GL更具优势。这项研究的重点是研究特殊活性基团对天然产物药理作用的影响，受到分子对接结果的启发。
