丹防胶囊对免疫性肝纤维化大鼠肝组织wnt1、β-catenin、DKK1表达的影响

Effects of danfang capsule on expressions of wnt1, β-catenin and DKK1 in rats with immunological hepatic fibrosis

目的探讨丹防胶囊对大鼠免疫性肝纤维化组织中wnt1、β-catenin及DKK1表达的影响及其可能机制。方法60只清洁级SD大鼠随机分为6组:正常组、模型组、阳性药物组、低丹组、中丹组、高丹组,每组10只。除正常组外其余各组腹腔注射猪血清制备大鼠免疫性肝纤维化模型,正常组腹腔注射等量生理盐水,2次/周。造模同时,低丹组、中丹组及高丹组分别给予丹防胶囊0.54、2.16、4.32g/kg灌胃,阳性药物组给予0.54g/kg复方鳖甲软肝片灌胃,正常组及模型组给予生理盐水灌胃,1次/d,共12周。12周末麻醉处死大鼠,行肝组织病理学检查,碱水解法检测肝组织羟脯氨酸(HYP)含量,免疫组织化学染色、Western blot、qPCR检测大鼠肝组织wnt1、β-catenin、DKK1的表达。结果与正常组比较,模型组肝纤维化明显,HYP含量和wnt1、β-catenin表达水平升高,DKK1表达水平降低(均P<0.05);与模型组比较,低丹组肝纤维化程度、HYP含量及wnt1、β-catenin和DKK1表达差异无统计学意义(P>0.05),中丹组、高丹组肝纤维化程度明显减轻,HYP含量和wnt1、β-catenin表达水平降低,DKK1表达水平升高(均P<0.05);与阳性药物组比较,低、中丹组肝纤维化严重,HYP含量和wnt1、β-catenin表达水平升高,DKK1表达水平降低(均P<0.05),高丹组与其差异无统计学意义(P>0.05);低、中、高丹组中肝纤维化程度依次减轻,HYP含量及wnt1、β-catenin表达水平依次降低,DKK1表达水平依次升高,组间多重比较差异均有统计学意义(均P<0.05)。结论丹防胶囊对大鼠免疫性肝纤维化起保护作用,并呈剂量相关性,其机制可能与上调肝组织DKK1表达,下调wnt1、β-catenin表达,从而抑制wnt/β-catenin通路激活有关。

Objective To investigate the effect of danfang (Dan) capsule on the expressions of wnt1,β-catenin and DKK1 in rat immune liver fibrosis and its possible mechanism. Methods Sixty clean grade SD rats were randomly divided into six groups: normal group, model group, low- Dan group, medium- Dan group, high- Dan group and positive drug group, 10 rats per group. In addition to the normal group, the rest groups were intraperitoneally injected with pig serum to prepare rat models of immune liver fibrosis, and the normal group was intraperitoneally injected with the same amount of normal saline twice per week. At the same time, different concentrations (0.54, 2.16, 4.32 g/kg) of danfang capsules were given by gavage to low-Dan group, medium-Dan group and high-Dan group. Compound turtle shell soft liver tablet (0.54 g/kg) was given by gavage to positive drug group, while the same amount of saline was given by gavage to normal group and model group once a day for 12 weeks. Rats were killed by anesthesia at the end of 12 weeks. Liver histopathological examination was performed. Alkaline hydrolysis was used to measure the content of hydroxyproline (HYP). The immunohistochemistry staining, Western blot assay and qPCR were used to measure the expressions of wnt1,β-catenin and DKK1. Results Compared with normal group, the hepatic fibrosis was serious in model group, with increased HYP content, wnt1 and β- catenin expressions and decreased DKK1 expression (all P<0.05). Compared with model group, there were no significant differences in liver fibrosis degree, HYP content, expressions of wnt1,β-catenin and DKK1 in the low-Dan group (P>0.05). The fibrosis degree was significantly reduced in medium-Dan group and high-Dan group, and the HYP content and the expressions of wnt1 and β-catenin were reduced, while the expression of DKK1 was increased (all P<0.05). Compared with the positive drug group, liver fibrosis was more severe in the low-Dan and medium-Dan groups, with increased HYP content, wnt1 and β-catenin expressions, and decreased DKK1 expression (all P<0.05). There were no significant differences in these indicators between positive drug group and high-Dan group (P>0.05). In the low-Dan,medium-Dan and high-dan groups, hepatic fibrosis was reduced in turn, HYP content, and expressions of wnt1 and β-catenin were decreased in turn, and DKK1 expression was increased in turn. There were significant differences among the groups in multiple comparisons (all P<0.05). Conclusion Danfang capsule has a certain intervention effect on immune liver fibrosis in rats, with the most significant effect at high dose. The mechanism may be related to the up-regulated expression of DKK1 and down-regulated expressions of wnt1 and β-catenin, thus inhibiting the activation of Wnt/β-catenin pathway.