茯苓酸对银屑病小鼠皮损组织IRS-1/ERK1/2通路及角质形成细胞增殖的影响

Effects of pachymic acid on IRS-1/ERK1/2 pathway and keratinocytes proliferation in skin damage tissues of psoriasis mice

目的探讨茯苓酸(PA)对银屑病小鼠皮损组织胰岛素受体底物(IRS)-1/细胞外调节蛋白激酶(ERK)1/2通路及角质形成细胞增殖的影响。方法将BALB/C雄性小鼠随机分为正常对照组、模型组、PA低、中、高(1.25、2.50、5.00 mg/kg)剂量组、阳性药物组(氨甲蝶呤,2 mg/kg),每组10只。除正常对照组外,其余各组于背部涂抹5%咪喹莫特乳膏建立银屑病模型;各组均在分组后当天开始给药,PA各剂量组及阳性药物组灌胃给予相应剂量的PA和氨甲蝶呤,正常对照组和模型组灌胃给予相应体积生理盐水,连续给药1 w,1次/d。各组末次给药24 h后,观察各组背部皮肤组织,并对银屑病皮损面积和严重程度指数(PASI)进行评分;取背部皮损组织,采用苏木素-伊红(HE)染色观察皮肤组织形态学改变;取血液和皮损组织,用酶联免疫吸附试验检测空腹胰岛素(FINS)、空腹血糖(FBG)水平及皮损组织炎性因子白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α、IL-17、IL-23含量,计算胰岛素抵抗指数(HOMA-IR);Western印迹检测皮损组织IRS-1、ERK1/2、磷酸化ERK1/2(p-ERK1/2)蛋白及角质形成细胞增殖、分化指标-增殖细胞核抗原(PCNA)和外皮蛋白(Involucrin)表达水平。结果正常对照组皮肤组织正常;与正常对照组相比,模型组可见背部皮肤红斑、鳞屑、增厚浸润等损伤及表皮层棘细胞层增厚、角质层角化不全、真皮层炎性细胞浸润等病理损伤程度、PASI评分、HOMA-IR及皮损组织IL-6、TNF-α、IL-17、IL-23含量、IRS-1、p-ERK1/2、PCNA、Involucrin蛋白表达水平明显升高(P<0.05);与模型组相比,PA低、中、高剂量组及阳性药物组背部皮肤损伤及皮损组织病理损伤程度均减轻、PASI评分、HOMA-IR及皮损组织IL-6、TNF-α、IL-17、IL-23含量、IRS-1、p-ERK1/2、PCNA、Involucrin蛋白表达水平明显降低,且PA各剂量组上述指标呈剂量依赖性降低(P<0.05);PA高剂量组与阳性药物组上述指标差异无统计学意义(P>0.05);各组皮损组织ERK1/2蛋白表达水平差异无统计学意义(P>0.05)。结论PA可抑制皮损组织IRS-1/ERK1/2通路激活,降低PCNA、Involucrin蛋白表达,抑制皮损组织角质形成细胞增殖和分化,改善银屑病模型小鼠胰岛素抵抗、炎症反应及病理损伤。

Objective To investigate the effects of pachymic acid(PA)on insulin receptor substrate(IRS)-1/extracellular regulated protein kinases(ERK)1/2 pathway and keratinocyte proliferation in the skin damage tissues of psoriasis mice.Methods BALB/C male mice were randomly divided into normal control group,model group,PA low,medium,high(1.25,2.50,5.00 mg/kg)dose groups and positive drug group(methotrexate,2 mg/kg),with 10 mice in each group.In addition to normal control group,the other groups were given 5%imiquimod cream on the back to establish psoriasis model;each group were administered on the day after grouping,the corresponding doses of PA and methotrexate were given by gavage to PA dose groups and positive drug group,and the corresponding volume of normal saline was given by gavage to the normal control group and model group,administrated for one week,once a day.24 h after the last administration,the skin tissues of the back in each group were observed,and the psoriasis area and severity index(PASI)score was conducted;back tissues were taken to observe the histomorphology of the skin by hematoxylin eosin(HE)staining;blood and skin damage tissues were taken to measure the levels of fasting insulin(FINS),fasting blood glucose(FBG)and the contents of inflammatory factor interleukin(IL)-6,tumor necrosis factor(TNF)-α,IL-17,IL-23 in skin damage tissues by enzyme-linked immunosorbent assay,and the insulin resistance index(HOMA-IR)was calculated;Western blot was used to detect the expression levels of IRS-1,ERK1/2,phosphorylation-ERK1/2(p-ERK1/2)protein,indicators of keratinocyte proliferation and differentiation-proliferating cell nuclear antigen(PCNA)and Involucrin in skin damage tissues.Results The skin tissues of normal control group was normal;compared with the normal control group,the injuries such as erythema,scaly,thickening and infiltration of the back skin,and the pathological damage such as thickening of the epidermal spinous cell layer,incomplete keratinization,and inflammatory cell infiltration in the dermis were observed,and the PASI score,HOMA-IR,the contents of IL-6,TNF-α,IL-17,IL-23,and the expression levels of IRS-1,p-ERK1/2,PCNA and Involucrin proteins in skin damage tissues were significantly increased in model group(P<0.05);compared with the model group,the degrees of skin damage and pathological damage of the back in the PA lower,middle and high dose groups and the positive drug group were reduced,the PASI score,HOMA-IR,the contents of IL-6,TNF-α,IL-17,IL-23,and the expression levels of IRS-1,p-ERK1/2,PCNA and Involucrin proteins in skin damage tissues were significantly decreased,and the above indexes of PA dose groups were decreased in a dose-dependent manner(P<0.05);while compared with PA high dose group,there was no significant difference in the above indexes in the positive drug group(P>0.05);there were no significant differences in the expression levels of ERK1/2 protein in skin damage tissues in each group(P>0.05).Conclusions PA could inhibit the activation of IRS-1/ERK1/2 pathway in skin damage tissues,decrease the protein expression of Involucrin and PC-NA,inhibit the proliferation and differentiation of keratinocytes in skin damage tissues,improve the insulin resistance,inflammatory response and pathological damage of psoriasis model mice.