矢志方激活Sirt1-Foxo1通路调控内质网应激相关蛋白改善高尿酸血症小鼠肾损伤

Shizhi Prescription regulating endoplasmic reticulum stress-related proteins to ameliorate renal injury in hyperuricemic mice by activating Sirt1-Foxo1 pathway

关键词:  矢志方  高尿酸血症  Sirt1-Foxo1通路  内质网应激

Key words:  Shizhi Prescription  hyperuricemia  Sirt1-Foxo1 pathway  endoplasmic reticulum stress

摘要:

[目的] 基于沉默信息调节因子1（Sirt1）-叉头转录因子1（Foxo1）通路观察矢志方对高尿酸血症（HUA）小鼠肾组织内质网应激（ERS）及下游分子增强子结合蛋白同源蛋白（Chop）、天冬氨酸特异性半胱氨酸蛋白酶（Caspase） 12表达的影响，探讨其作用机制。[方法] 32只SPF级雄性BALB/c小鼠随机分为正常组、模型组、非布司他组和矢志方组，每组8只。正常组给予生理盐水灌胃，其余各组予氧嗪酸钾250 mg/kg灌胃制备HUA小鼠模型。非布司他组和矢志方组分别按6 mg/kg、562.5 mg/kg灌胃相应药物，正常组和模型组予相同体积生理盐水灌胃，连续2周。体外实验培养人肾小管上皮细胞（HK2），分为正常组、模型组、矢志方组，饥饿24 h后予200 μg/mL尿酸和300 μg /mL矢志方干预24 h。苏木精-伊红（HE）染色和Masson染色观察小鼠肾脏组织病理变化，蛋白免疫印迹法（Western blot）和免疫组化染色检测小鼠肾组织Sirt1、内质网跨膜蛋白肌醇酶1α（IRE1α）、Foxo1、Caspase 12、Chop表达，Western blot和免疫荧光染色检测HK2细胞Sirt1、IRE1α、Foxo1、Caspase 12、Chop表达。[结果] HE染色和Masson染色结果显示：与正常组比较，模型组小鼠肾小管管壁变薄，管腔扩张，炎性细胞浸润，大量蓝色胶原纤维沉积。与模型组比较，矢志方组小鼠肾小管管壁变薄、管腔扩张、蓝色胶原纤维集聚程度减少。Western blot结果显示：模型组较正常组相比肾组织和HK2细胞IRE1α、Foxo1、Caspase 12、Chop表达显著升高（P＜0.01，P＜0.001），Sirt1蛋白表达显著降低（P＜0.001），用药组较模型组相比肾组织和HK2细胞IRE1α、Foxo1、Caspase 12、Chop蛋白表达显著降低（P＜0.05，P＜0.001），Sirt1蛋白表达显著升高（P＜0.001）。免疫组化染色和免疫荧光染色结果与Western blot结果一致。[结论] 矢志方减轻肾脏病理损伤的机制可能与抑制 HUA 小鼠肾组织ERS、激活Sirt1-Foxo1通路表达有关。

Abstract:

[Objective] To observed the effects of Shizhi Prescription（SZF） on endoplasmic reticulum stress-related proteins and downstream molecules Chop/Caspase12 in the kidney tissues of hyperuricemic mice to explore its mechanism based on the Sirt1-Foxo1 pathway. [Methods] Thirty-two SPF male BALB/c mice were randomly divided into normal group，model group，febuxostat group and SZF group. The normal group was gavaged with saline，the other groups were gavaged with 250 mg/kg potassium oxyzincate to prepare the HUA mouse model. The febuxostat and SZF groups were given 6 mg/kg and 562.5 mg/kg gavage respectively，the normal and model groups were given the same volume of saline for 2 weeks. In vitro experiments were performed with HK2 cells. Divided into normal group，model group and SZF group. The 200 μg/mL uric acid and 300 μg/mL SZF were treated after 24 h of starvation to intervene for 24 h. Histopathological changes of mouse kidney were observed by HE and Masson staining. Sirt1，IRE1α，Foxo1，Caspase12 and Chop were detected by Western blot and immunohistochemical staining. Western blot and immunofluorescence staining to detect Sirt1，IRE1α，Foxo1，Caspase12 and Chop expression in HK2 cells. [Results] The results of HE staining and Masson staining showed that the tubular wall was thinner，the lumen was dilated，inflammatory cells were infiltrated，and the interstitial fibrous tissue was proliferated in the model group compared with the normal group. Western blot showed that the expression of IRE1α，Foxo1，Caspase12 and Chop was significantly higher in the model group compared with the normal group（P＜0.01 or P＜0.001）. Sirt1 protein expression was significantly decreased（P＜0.001）. Compared with the model group. IRE1α，Foxo1，Caspase12，Chop protein expression was significantly decreased（P＜0.05 or P＜0.001） and Sirt1 protein expression was significantly increased（P＜0.001） in kidney tissues and HK2 cells in the drug-administered group. Immunohistochemical staining and immunofluorescence staining results were consistent with Western blot results. [Conclusion] The mechanism of alleviating renal pathological injury by SZF may be related to the inhibition of endoplasmic reticulum stress and activation of Sirt1-Foxo1 pathway in the renal tissue of HUA mice.