健脾解毒汤对脾虚湿盛证银屑病样大鼠皮肤屏障功能调控机制研究

Mechanism of Jianpi Jiedu decoction in the regulation of skin barrier function of rats with psoriasis vulgaris of syndrome of spleen deficiency and excess dampness

目的 探讨细胞外信号调节激酶/p38丝裂原活化蛋白激酶(ERK/p38MAPK)信号通路在健脾解毒汤调控脾虚湿盛证银屑病大鼠皮肤组织水通道蛋白(AQP3)表达中的作用。方法按照随机数字表法,将144只SPF级雄性SD大鼠分为空白组、模型组、甲氨蝶呤组、健脾解毒汤低剂量组、健脾解毒汤中剂量组、健脾解毒汤高剂量组、健脾解毒汤低剂量+ERK抑制剂组、健脾解毒汤中剂量+ERK抑制剂组、健脾解毒汤高剂量+ERK抑制剂组、健脾解毒汤低剂量+MAPK抑制剂组、健脾解毒汤中剂量+MAPK抑制剂组、健脾解毒汤高剂量+MAPK抑制剂组,每组12只。除空白组外,其余组大鼠采用咪喹莫特外涂和大黄配方颗粒剂灌胃方法构建脾虚湿盛证银屑病样皮损复合模型。造模成功后,甲氨蝶呤组给予甲氨碟呤片0.97 mg/kg灌胃,健脾解毒汤低、中、高剂量组分别给予2.32 g/kg、4.64 g/kg、9.26 g/kg健脾解毒汤灌胃,健脾解毒汤低、中、高剂量+ERK抑制剂各组均给予ERK抑制剂U0126腹腔注射和相应剂量的健脾解毒汤灌胃,健脾解毒汤低、中、高剂量+MAPK抑制剂各组均给予MAPK抑制剂SB203580腹腔注射和相应剂量的健脾解毒汤灌胃,空白组和模型组给予等量的生理盐水灌胃和腹腔注射,均1次/d,连续14 d。灌胃结束后,观察大鼠皮肤组织病理学形态,Western blot法检测皮肤组织中AQP3、ERK1/2、p-ERK1/2、p38 MAPK、p-p38 MAPK蛋白表达情况。结果 模型组皮损部位表皮增生肥厚,表皮内及角质层内较多中性粒细胞浸润,真皮浅层内血管扩张,慢性炎细胞散在或灶状浸润;各药物组皮肤组织病变程度均较模型组轻,健脾解毒汤高剂量组、健脾解毒汤高剂量+ERK抑制剂组、健脾解毒汤高剂量+MAPK抑制剂组病变程度更轻。与空白组比较,模型组大鼠皮肤组织中AQP3蛋白相对表达量明显降低(P<0.05),p-ERK1/2、p-p38 MAPK蛋白相对表达量均明显升高(P均<0.05);与模型组比较,甲氨蝶呤组、健脾解毒汤各剂量组、健脾解毒汤高剂量+ERK抑制剂组、健脾解毒汤高剂量+MAPK抑制剂组AQP3蛋白相对表达量均明显升高(P均<0.05),p-ERK1/2、p-p38 MAPK蛋白相对表达量均明显降低(P均<0.05);且健脾解毒汤高剂量+ERK抑制剂组、健脾解毒汤高剂量+MAPK抑制剂组AQP3蛋白相对表达量较其他药物组升高更明显(P均<0.05),p-p38 MAPK蛋白相对表达量较其他药物组降低更明显(P均<0.05),p-ERK1/2蛋白相对表达量较甲氨蝶呤组和健脾解毒汤低、中剂量组降低更明显(P均<0.05)。结论 健脾解毒汤可能是通过抑制ERK/p38 MAPK信号通路的激活,上调AQP3蛋白表达,从而使体内水液分布均衡,恢复皮肤屏障功能而发挥治疗银屑病的作用。

Objective It is to investigate the effects of extracellular-signalregulated protein kinase(ERK)/p38 mitogen activated protein kinase(MAPK)signal pathway in the regulation of expression of aquaporin 3(AQP3)by Jianpi Jiedu decoction in rats with psoriasis vulgaris of syndrome of spleen deficiency and excess dampness.Methods One hundred and forty four SD rats of SPF grade were randomly divided into blank group,modle group,methotrexate(MTX)group,Jianpi Jiedu decoction low dose group,Jianpi Jiedu decoction median dose group,Jianpi Jiedu decoction hige dose group,Jianpi Jiedu decoction low dose+ERK inhibitor group,Jianpi Jiedu decoction median dose+ERK inhibitor group,Jianpi Jiedu decoction high dose+ERK inhibitor group,Jianpi Jiedu decoction low dose+MAPK inhibitor group,Jianpi Jiedu decoction median dose+MAPK inhibitor group,Jianpi Jiedu decoction high dose+MAPK inhibitor group,with 12 rats in each group.Except for the blank group,the rats in the other groups were given imiquimod ointment by external application and rhubarb formula granule by garage to establish compound models of psoriasis vulgaris like of syndrome of spleen deficiency and excess dampness.After successful modeling,the MTX group was given 0.97 mg/kg MTX tablets by gavage,the low,medium,and high dose groups of Jianpi Jiedu decoction were respectively given 2.32 g/kg,4.64 g/kg,and 9.26 g/kg Jianpi Jiedu decoction by gavage,the groups of low,medium,and high dose Jianpi Jiedu decoction+ERK inhibitor were given the ERK inhibitor U0126 by intraperitoneal injection and the corresponding dose of Jianpi Jiedu decoction by gavage,the groups of low,medium,and high dose Jianpi Jiedu decoction+MAPK inhibitor were given the MAPK inhibitor SB203580 by intraperitoneal injection and the corresponding dose of Jianpi Jiedu decoction by gavage,the blank group and model group were given the same amount of normal saline by gavage and intraperitoneal injection,all of them were treated once per day,continuously treated for 14 days.Results In the model group,epidermal hyperplasia and hypertrophy at the lesion site were found,with more neutrophilic infiltration in the epidermis and stratum corneum,vasodilatation in the superficial dermis,and chronic scattered or focal infiltration of inflammatory cells.The degree of skin tissue lesions in all treatment groups was lighter than that in the model group,and the degree of lesions was more lighter in the Jianpi Jiedu decoction high dose group,Jianpi Jiedu decoction high dose+ERK inhibitor group,Jianpi Jiedu decoction high dose+MAPK inhibitor group.Compared with the blank group,the relative expression of AQP3 protein in the skin tissues of rats was significantly lower(P<0.05),and the relative expressions of p-ERK1/2 and p-p38 MAPK protein were significantly higher in the model group(both P<0.05);compared with the model group,the relative expression of AQP3 protein in the MTX group,each dose groups of Jianpi Jiedu decoction,Jianpi Jiedu decoction high dose+ERK inhibitor group,Jianpi Jiedu decoction high dose+MAPK inhibitor group were all significantly increased(all P<0.05),and the relative expressions of p-ERK1/2 and p-p38 MAPK protein were significantly decreased(all P<0.05);the increase of relative expression of AQP3 protein and the decrease of relative expression of p38 MAPK protein in the Jianpi Jiedu decoction high dose+ERK inhibitor group,Jianpi Jiedu decoction high dose+MAPK inhibitor group were more significantly than that in the other treatment group(P<0.05),the decrease of relative expression of p-ERK1/2 protein was more significantly than that in the MTX group,and low,medium dose groups of Jianpi Jiedu decoction(all P<0.05).Conclusion Jianpi Jiedu decoction can up-regulate the expression of AQP3 in skin tissue via inhibiting the activation of the ERK/p38MAPK signaling pathway,thus to balance the water and liquid distribution in body,restore the skin barrier function in the treatment of psoriasis vulgaris.