红景天苷对C57BL/6衰老小鼠肝保护的作用机制

Hepatoprotective Effect and Mechanism of Salidroside in Senile C57BL/6 Mice

目的探讨红景天苷(Salidroside,Sal)对C57BL/6衰老小鼠的肝保护的作用机制。方法在50只SPF级雄性C57BL/6小鼠中随机选取10只正常饲养至3个月龄作为对照组,其余40只C57BL/6小鼠饲养至13个月龄作为早期衰老小鼠,随机分为衰老组、阳性药环黄芪醇(CAG)组、红景天苷低剂量(Sal-L)组、红景天苷高剂量(Sal-H)组。对照组和衰老组小鼠分别给予等量生理盐水10 ml/kg灌胃,阳性药物组给予CAG 20 mg/kg灌胃,Sal-L组和Sal-H组分别给予红景天苷25 mg/kg和100 mg/kg灌胃,1次/d,疗程60 d。治疗结束后,进行老化度评分。比色法检测小鼠血清单胺氧化酶(Monoamine oxidase,MAO)和超氧化物歧化酶(Superoxide dismutase,SOD)的活性,硫代巴比妥酸法检测脂质过氧化物丙二醛(Monoamine oxidase,MDA)含量,荧光探针法检测活性氧(Reactive oxygenspecies,ROS)含量。Masson染色法观察小鼠肝组织纤维化情况、醛品红染色法观察小鼠肝组织脂褐素沉积情况。结果与对照组比较,衰老组小鼠老化度评分明显升高,差异有统计学意义(P<0.01)。与衰老组比较,CAG组、Sal-L组、Sal-H组小鼠老化度评分均明显降低,差异有统计学意义(P<0.01)。与对照组比较,衰老组小鼠血清SOD活性明显下降,差异有统计学意义(P<0.01)。与衰老组比较,CAG组、Sal-H组小鼠血清SOD活性明显升高,差异有统计学意义(P<0.05)。与对照组比较,衰老组小鼠血清ROS水平明显升高,差异有统计学意义(P<0.01)。与衰老组比较,CAG组、Sal-L组、Sal-H组小鼠血清ROS水平均明显降低,差异有统计学意义(P<0.01)。Sal-H组小鼠ROS水平明显低于Sal-L组,差异有统计学意义(P<0.05)。与对照组比较,衰老组小鼠血清MAO活性明显升高,差异有统计学意义(P<0.01)。与衰老组比较,CAG组、Sal-L组、Sal-H组小鼠血清MAO活性均明显降低,差异有统计学意义(P<0.01)。与对照组比较,衰老组小鼠血清MDA含量明显升高,差异有统计学意义(P<0.01)。与衰老组比较,CAG组、Sal-L组、Sal-H组小鼠血清MDA含量均明显降低,差异有统计学意义(P<0.01)。Masson染色结果显示,对照组小鼠肝细胞清晰可见,呈红色的纤维排列整齐,无纤维结缔组织增生。衰老组染色为蓝色的胶原纤维较多,肝细胞排列紊乱,胶原纤维增生,CVF较对照组明显升高,差异有统计学意义(P<0.01)。与衰老组比较,Sal-H组和CAG组小鼠肝纤维化改善,蓝色胶原纤维增生减少,CVF明显降低,差异有统计学意义(P<0.05,P<0.01)。醛品红染色结果显示,对照组小鼠肝细胞轮廓清晰,排列整齐,胞浆清亮。衰老组肝细胞胞体萎缩,排列不规则,胞浆内出现大量深紫色的脂褐素颗粒,CVF较对照组明显升高,差异有统计学意义(P<0.01)。与衰老组比较,CAG组、Sal-H组小鼠肝组织脂褐素沉积情况有所改善,胞浆紫色色素颗粒减少,CVF明显降低,差异有统计学意义(P<0.05)。结论红景天苷可有效降低衰老小鼠老化度评分、肝组织ROS水平,减轻衰老小鼠肝组织氧化应激损伤,改善衰老小鼠肝纤维化和肝组织中脂褐素沉积,表明红景天苷可通过抗氧化应激来发挥其抗衰老作用。

Objective To investigate the hepatoprotective effect and mechanism of salidroside in senile C57BL/6 mice.Methods From 50 SPF-grade C57BL/6 male mice,10 mice were randomly selected and reared to 3 months old as the young control group.The other 40 mice were reared to 13 months as early aging mice and then randomized into aging,positive drug(cycloastragenol,CAG,20 mg/kg),and salidroside low-dose(Sal-L,25 mg/kg)and high-dose(Sal-H,100 mg/kg)groups.The mice in young control group and aging group were administrated with the same amount of normal saline at 10 mL/kg by gavage,and those in other groups with corresponding drugs by gavage,respectively,once a day for 60 days.After administration,the aging degrees of mice were evaluated.The colorimetric method was used to determine the activities of monoamine oxidase(MAO)and superoxide dismutase(SOD)in the serum.The levels of malondialdehyde(MDA)and reactive oxygen species(ROS)in the serum were measured by thiobarbituric acid assay and fluorescent probe assay,respectively.The fibrosis of mouse liver tissue was observed by Masson staining and lipofuscin deposition by aldehyde fuchsin staining.Results The aging group had higher aging degree than the control,CAG,Sal-L,and Sal-H groups(P<0.01).Compared with the control group,the aging group showed decreased SOD activity(P<0.01).Compared with the aging group,the CAG and Sal-H group had increased SOD activity(P<0.05).The aging group had higher ROS level than the control,CAG,Sal-L,and Sal-H groups(P<0.01),and the Sal-H group had lower ROS level than the Sal-L group(P<0.05).The aging group had higher MAO activity and MDA level than the control,CAG,Sal-L,and Sal-H groups(P<0.01).The Masson staining results showed that the hepatocytes in the control group were clear,with red fibers arranged neatly and no fibrous connective tissue proliferation.The aging group had increased collagen fibers stained blue,disordered arrangement of hepatocytes,collagen fiber hyperplasia,and increased cell volume fraction(CVF)compared with the control group(P<0.01).Compared with the aging group,Sal-H and CAG mitigated liver fibrosis and reduced blue collagen fiber hyperplasia and CVF(P<0.05,P<0.01).The results of aldehyde fuchsin staining showed that the hepatocytes in the control group had clear boundary,neat arrangement,and clear cytoplasm.The hepatocytes in the aging group showed atrophy,disordered arrangement,massive dark purple lipofuscin particles in the cytoplasm,and increased CVF compared with the control group(P<0.01).Compared with the aging group,Sal-H and CAG mitigated the deposition of lipofuscin in the liver tissue and reduced the purple pigment granules in cytoplasm and CVF(P<0.05).Conclusion Salidroside can decrease the aging degree,lower the ROS level to alleviate the oxidative stress,and mitigate the liver fibrosis and lipofuscin deposition in liver tissue of aging mice,thus playing an anti-aging role.