齐墩果酸通过调控STAT3通路改善咪喹莫特诱导的银屑病样小鼠皮损表现

摘要 目的：观察齐墩果酸对咪喹莫特诱导的银屑病样小鼠的皮损及STAT3通路的干预作用。方法：72只BALB/c雄性小鼠，按随机数字表法随机分为空白组、模型组、甲氨蝶呤组、齐墩果酸低、中、高剂量组，每组12只，于背部备皮。除空白组外，其余各组小鼠每日于背部备皮区域涂抹5%咪喹莫特乳膏诱导银屑病样皮损；甲氨蝶呤组及齐墩果酸各浓度组每日灌胃对应药物0.2 mL/只；每日观察皮损状态并拍照记录，每日对小鼠皮损面积及严重程度（Psoriasis Area and Severity Index，PASI）进行评分；造模第4、7天取小鼠皮损区域皮肤，免疫印迹法检测皮损中STAT3信号通路中STAT3磷酸化表达情况；第7天称量小鼠体重及脾重，计算脾指数，同时取小鼠皮损区域皮肤；苏木素-伊红（HE）染色观察皮损组织病理学改变，并测量表皮厚度；免疫组织化学法及免疫组织荧光法检测皮损中CD3⁺、CD4⁺、CD8⁺T细胞浸润情况及Ki67表达情况；实时荧光定量PCR法检测皮损中白介素-17（Interleukin-17，IL-17）mRNA相对表达情况。结果：与空白组相比，造模后模型组小鼠背部皮肤出现红斑、鳞屑、浸润性皮损，第4天及第7天PASI评分升高（P<0.01）；第7天模型组表皮厚度明显增加，体重下降，脾指数上升，皮损中Ki67、CD3⁺、CD4+⁺、CD8⁺T细胞表达量升高，皮损中IL-17的mRNA相对表达量升高（均P<0.01）；第4及第7天，模型组小鼠皮损中p-STAT3的表达量均明显升高（P<0.05，P<0.01）；与模型组相比，第4天各治疗组在PASI评分略降低（P>0.05），第7天各治疗组PASI评分明显降低（均P<0.01）；第7天齐墩果酸各剂量组小鼠体重略有上升（P>0.05）；甲氨蝶呤组小鼠脾指数明显下降（P<0.01），齐墩果酸低、中剂量组小鼠脾指数略下降（P>0.05）；各治疗组小鼠表皮厚度较模型组明显下降（P<0.01或P<0.05），小鼠皮肤中Ki-67表达量、CD4⁺、CD8⁺T细胞表达量均明显降低（均P<0.01）；甲氨蝶呤组、齐墩果酸低、中、高剂量组小鼠皮损中CD3⁺T细胞表达量均下降（P<0.05，P>0.05，P<0.01，P<0.01），其中在齐墩果酸各剂量组中，中剂量组在脾指数下降、表皮厚度变薄、CD3⁺、CD4⁺、CD8⁺T细胞减少方面均略优于低、高剂量组（P>0.05）；甲氨蝶呤组及齐墩果酸中剂量组小鼠皮损中IL-17的mRNA水平较模型组明显下降（P<0.01，P<0.05）；造模给药第4天，甲氨蝶呤组小鼠皮损中p-STAT3水平较模型组明显下降（P<0.05），齐墩果酸中剂量组略下降（P>0.05）；造模给药第7天，两个治疗组小鼠皮损中p-STAT3水平较模型组均明显下降（均P<0.01）。结论：齐墩果酸低、中、高剂量均可以改善咪喹莫特诱导的小鼠银屑病样皮损的严重程度，减轻炎性细胞浸润，中剂量组疗效较好，其机制可能通过抑制STAT3通路中p-STAT3的表达从而降低了IL-17的含量，且药效随作用时间的延长而逐渐增加。

Oleanolic Acid Ameliorates Imiquimod-induced Psoriatic Lesions in Mice by Regulating STAT3 Pathway

ABSTRACT Objective: To observe the effects of oleanolic acid on psoriatic lesions and STAT3 pathway in imiquimod-induced mice. Methods: 72 BALB/c male mice were randomly divided into control group, model group, methotrexate group, along with oleanolic acid low-dose group, medium-dose group and high-dose group according to random number table method, with 12 mice in each group. Except the control group, each group of mice was topical applicated with 5% imiquimod cream on the back skin to induce psoriasis-like lesions. Methotrexate group and oleanolic acid groups were administered intragastrically with 0.2 mL of corresponding drugs per mouse. Observe and take photos daily to record the changes of the skin lesion. The psoriasis area and severity index (PASI) of mice were scored daily. On day 4 and 7 of modeling, the skin lesions of mice were taken and the phosphorylation of STAT3 was detected by western blotting. On the 7th day, the body weight and spleen weight of mice were weighed to calculate the spleen index, and the skin in the lesions area of mice was taken. Hematoxylin-eosin (HE) staining was used to observe the histopathological changes and measure the epidermal thickness. CD3⁺, CD4⁺, CD8⁺T cell infiltration and Ki67 expression were detected by immunohistochemistry and immunofluorescence. The relative expression of interleukin-17(IL-17) mRNA in skin lesions was detected by real-time quantitative PCR. Results: Compared with blank group, the back skin of mice in model group showed erythema, scales and infiltrating skin lesions after modeling, and the PASI score increased on day 4 and 7(P<0.01). On day 7, the epidermal thickness, body weight, spleen index, Ki67, CD3⁺, CD4⁺, CD8⁺T cell expression in the skin lesion increased significantly, and the mRNA relative expression of IL-17 in the skin lesion increased (all P<0.01). On day 4 and 7, the expression of p-STAT3 in the skin lesions of model group was increased significantly (P<0.05, P<0.01). Compared with the model group, the PASI score in each treatment group was lower at the 4th day(P>0.05), the PASI score was decreased significantly at the 7th day(P<0.01). On day 7, the body weight of mice in each dose of oleanolic acid groups increased slightly (P>0.05). The spleen index of mice in methotrexate group decreased significantly(P<0.01), spleen index of mice in low and medium dose of oleanolic acid groups decreased slightly (P>0.05). Compared with model group, epidermal thickness of mice in treatment groups were decreased significantly (P<0.01 or P<0.05), and the expression levels of Ki-67, CD4⁺ and CD8⁺T cells in skin were decreased significantly (all P<0.01). The expression of CD3⁺T cells in the skin lesions of mice in methotrexate group and three doses of oleanolic acid groups were decreased(P<0.05, P>0.05, P<0.01, P<0.01). The mRNA levels of IL-17 in the skin lesions of methotrexate group and oleanolic acid medium-dose group were significantly lower than that of model group(P<0.01, P<0.05). On day 4, the level of p-STAT3 in skin lesions in methotrexate group was significantly lower than that in model group(P<0.05), and that in oleanolic acid medium-dose group was slightly lower(P>0.05). On the 7th day, p-STAT3 level in skin lesions of mice in both treatment groups was significantly lower than that in model group(P<0.01). Conclusion: The three doses of oleanolic acid can improve the severity of imiquimod-induced psoriasis lesions and the expression of inflammatory cells. Among them, the medium does of oleanolic acid has a better effect. Inhibition of STAT3 signaling activation and decrease of IL-17 expression may be one of the mechanisms for its better effect. Moreover, the pesticide effect increases gradually with the prolongation of time.