西替利嗪通过TGF-β1/Smad3信号通路调控小鼠硬皮病皮肤组织纤维化研究

Cetirizine regulates scleroderma skin fibrosis in mice via the TGF-β1/Smad3 signaling pathway

目的:探讨西替利嗪对系统性硬皮病(systemic sclerosis,SSc)小鼠皮肤组织纤维化的影响及其作用机制。方法:将32只BALB/C小鼠随机分为空白组、模型组、西替利嗪低剂量组、西替利嗪高剂量组,每组8只。空白组背部注射生理盐水,其余3组均背部注射博来霉素制备SSc小鼠模型,1次/d,连续注射28 d,同时给予空白组与模型组生理盐水灌胃,西替利嗪低、高剂量组分别按照2、5 mg/kg灌胃,连续给药28 d。取各组背部注射区皮肤组织检测真皮厚度,进行苏木精-伊红染色(HE)和马松染色(Masson),样本水解法检测皮肤组织中羟脯氨酸(HYP)含量,免疫组织化学法检测皮肤组织α-平滑肌肌动蛋白(α-SMA)表达,酶联免疫吸附法(ELISA)检测血清白细胞介素(IL-6、IL-10)与转化生长因子(TGF-α、TGF-β1)的含量,荧光定量PCR(qRT-PCR)检测Ⅰ型胶原α1(COL1A1)、Ⅲ型胶原α1(COL3A1)、Smad同源物3(Smad3)、TGF-β1 mRNA的表达水平,蛋白印迹技术(Western blot)检测COL1A1、COL3A1、p-Smad3蛋白表达水平。结果:与空白组比较,模型组真皮厚度和HYP含量均增加,皮肤组织病变与纤维化程度较重,皮肤组织中α-SMA阳性表达强度增加,血清中IL-6、IL-10、TGF-α、TGF-β1的含量上升,皮肤组织中COL1A1、COL3A1、Smad3、TGF-β1 mRNA表达水平升高,COL1A1、COL3A1、p-Smad3蛋白表达量增加,差异均具有统计学意义(P<0.05)。与模型组比较,西替利嗪低、高剂量组真皮厚度和HYP含量均降低,皮肤组织病变与纤维化程度有所改善,皮肤组织中α-SMA阳性表达减弱,血清中IL-6、IL-10、TGF-α、TGF-β1的含量降低,皮肤组织中COL1A1、COL3A1、Smad3、TGF-β1 mRNA表达水平下降,COL1A1、COL3A1、p-Smad3蛋白表达量减少,且高剂量组效果更好,差异均具有统计学意义(P<0.05)。结论:西替利嗪可改善SSc小鼠皮肤组织纤维化程度,并减轻免疫炎症反应,其作用机制与TGF-β1/Smad3信号通路有关。

Objective To investigate the effect of cetirizine on the fibrosis of skin tissue in systemic sclerosis(SSc)mice and its mechanism of action.Methods Totally 32 BALB/C mice were randomly divided into a blank group,a model group,a cetirizine low-dose group,and a cetirizine high-dose group with 8 mice in each group.The blank group was injected with normal saline on the back,and the other three groups were injected with bleomycin on the back to prepare SSc mouse models.The mice were injected once a day for 28 consecutive days,while the normal group and the model group were given saline.The dose group was administrated intragastrically at 2 mg/kg and 5 mg/kg,respectively,for 28 consecutive days.The thickness of the dermis was detected by taking the skin tissue in the back injection area of each group with Hematoxylin-eosin staining(HE)and Masson staining.Sample hydrolysis method was used to detect hydroxyproline(HYP)content in skin tissue.Immunohistochemical detection ofα-smooth muscle actin(α-SMA)expression in skin tissues as well as Enzyme-linked immunosorbent assay(ELISA)to detect serum interleukin(IL-6,IL-10)and transforming growth factor(TGF-αand TGF-β1)were conducted.Quantitative real-time PCR(qRT-PCR)was used to detect the expression levels of collagen type I(COL1A1),typeⅢcollagen(COL3A1),Smad homolog 3(Smad3),and TGF-β1 mRNA.Western blot was used to detect the expression levels of COL1A1,COL3A1 and p-Smad3.Results Compared with the blank group,the dermis thickness and HYP content of the model group increased,the skin tissue lesions and fibrosis were more severe,theα-SMA positive expression intensity in the skin tissue was higher,and the serum IL-6,IL-10,TGF-α,TGF-β1 content increased,COL1A1,COL3A1,Smad3,TGF-β1 mRNA expression levels increased in skin tissues,COL1A1,COL3A1,and p-Smad3 protein expression increased with statistically significant differences(P<0.05).Compared with the model group,the dermal thickness and HYP content of the low and high dose cetirizine groups reduced,the degree of skin tissue lesions and fibrosis was improved,the expression ofα-SMA in skin tissues was weakened,the levels of IL-6,IL-10,TGF-α,TGF-β1 in serum,the expression levels of COL1A1,COL3A1,Smad3 and TGF-β1 in skin tissues and the expression levels of COL1A1,COL3A1,and p-Smad3 proteins reduced,and the decrease in the high-dose group was more significant with statistically significant differences(P<0.05).ConclusionCetirizine can improve the degree of fibrosis of skin tissue in SSc mice and reduce the immune inflammation response.The mechanism of action is related to the TGF-β1/Smad3 signaling pathway.