白芍总苷对硬皮病小鼠的治疗作用及机制研究

目的 通过给予硬皮病小鼠白芍总苷干预治疗,观察小鼠皮肤硬化的改善情况,探讨白芍总苷对硬皮病小鼠的治疗作用及可能机制.方法 将BALB/c小鼠随机分为A、B、C3组,分别给予皮下注射磷酸盐缓冲液(A组),博来霉素( B、C组).4周后,取皮损做病理切片、HE染色,检测羟脯氨酸含量,验证硬皮病模型是否成功.之后,A、B组以生理盐水灌胃,C组白芍总苷灌胃;4周后,检测皮损处羟脯氨酸、TGF-β1和Smad7的表达.结果 1)B、C组注射4周后,注射区皮肤明显硬化、脱毛.组织病理、HE染色及羟脯氨酸含量检测,证实硬皮病模型成功建立.2)实验8周后,组织HE染色提示: B组真皮层明显增厚,胶原纤维明显增多增粗,排列致密;C组较B组,其真皮层变薄,胶原纤维减少,排列疏松;羟脯氨酸含量比较:C组(313.556±40.660)明显低于B组(419.850±52.530),差异有统计学意义(P<0.05);而C组(313.556±40.660)稍高于A组(296.909±31.356),差异无统计学意义(P>0.05);免疫组化法测定TGF-β1和P-Smad7的变化:在A组小鼠皮损组织中,均呈弱阳性表达,在B、C组小鼠均呈强阳性表达;B组(1 198.080±376.234,763.500±287.640)的表达均高于A组(275.333±76.255,157.070±49.282)(P <0.05);C组中TGF-β1(836.571±313.656)明显低于B组(1 198.080±376.234),而C组中P-Smad7(1 337.494±405.052)明显高于B组(763.500±287.640),差异有统计学意义(P<0.05).结论 1)在BALB/c小鼠背部皮下注射博来霉素建立硬皮病模型的方法切实可行;2)白芍总苷可以有效缓解硬皮病皮肤硬化程度,其机制之一可能是通过上调Samd7的表达、抑制TGF-β1的表达,从而减轻皮肤的纤维化.

A Study on the Therapeutic Effect of Total Glucosides of Paeony on the Mice with Scleroderma and Its Mechanisms

Objective To explore the therapeutic effect of total glucosides of paeony (TGP) on the mice with scleroderma by treating them with TGP and observing the changes of their skin and investigate its mechanism.Methods The BALB/c mice were randomly divided into three groups including Group A injected subcutaneously with phosphate buffered saline (PBS), Group B treated with Bleomycin (BLM), and Group C treated with both BLM and TGP.The pathological section was made with skin lesions and the HE staining was performed to detect the hydroxyproline and confirm the establishment of the mouse models with scleroderma after 4 weeks.Then the mice of Group A and Group B were given normal saline by gavage, while the mice of Group C were treated with TGP by means of intragastric administration.The expression levels of hydroxyproline, TGF-β1 and Smad7 in the skin lesions were assayed after 4 weeks.Results Firstly, the mice of Group B and Group C had the symptoms of hardening and unhairing in the injection zone of skin after 4 weeks.The pathological section, HE staining and detection of hydroxyproline content confirmed the successful establishment of the mouse models with scleroderma.Secondly, The results of HE staining after 8 weeks showed that the dermis was thickened obviously, the collagen fiber was increased and thickened, and the arrangement was compact in Group B.Compared with Group B, Group C showed thinner dermis, less collagen fibers, and looser arrangement.The content of hydroxyproline of Group C (313.556±40.660) was significantly lower than that of Group B (419.850±52.530) (P<0.05), while it was a bit higher than that of Group A (296.909±31.356), and the difference was not statistically significant (P>0.05).The level changes of TGF-β1 and Smad7 in the immunohistochemical assay showed weak positive expression in Group A and strong positive expression in Group B and Group C.The expression of group B (1 198.080±376.234,763.500±287.640) was significantly higher than that of group A (275.333±76.255, 157.070±49.282) (P<0.05).The TGF-β1 expression of Group C (836.571±313.656) was significantly lower than that of Group B (1 198.080±376.234)(P<0.05), while the Smad7 expression of Group C (1337.494±405.052) was significantly higher than that of Group B (763.500±287.640) (P<0.05).Conclusion Firstly, The method of establishing the mouse model with scleroderma is feasible by the subcutaneous injection of bleomycin in the dorsal skin of BALB/c mice.Secondly, TGP can effectively alleviate the skin sclerosis of the mice with scleroderma and reduce the skin fibrosis by upregulating the Smad7 expression and inhibiting the TGF-β1 expression, which may be one of the mechanisms.