基于“通肾络、益脾肾”治法研究足细胞凋亡及自噬

Apoptosis and autophagy of podocytes studied based on “tonifying kidney collaterals and invigorating spleen and kidney”

关键词:  糖尿病肾病  通络益肾方  足细胞  凋亡  自噬  相互关系

Key words:  diabetic nephropathy  Tongluo Yishen Decoction  podocytes  apoptosis  autophagy  relationship

摘要:

[目的] 基于中医"通肾络、益脾肾"治法，探讨通络益肾方对体外高糖培养的小鼠肾小球足细胞自噬和凋亡蛋白SIRT1、BNIP3、P62、Bax表达的调控影响。[方法] 成熟无特定病原体（SPF）级SD雄性大鼠40只，随机分为正常组、中药高、中、低剂量组各10只。按照人与大鼠体表面积折算方法计算出大鼠所需中药灌胃浓度：高剂量浓度4.76 g/mL、中剂量浓度2.38 g/mL、低剂量浓度1.19 g/mL，灌胃10 d取含药血清备用。足细胞分6组，正常组5.6 mmol/L葡萄糖+10%正常大鼠血清、高糖组30mmol/L葡萄糖+10%正常大鼠血清、通络益肾方含药血清高、中、低干预组30 mmol/L葡萄糖+10%高、中、低剂量大鼠血清、高渗组甘露醇24.5 mmol/L+10%正常大鼠血清。细胞培养48 h后收集，Hoechst33342荧光染色，观察各组足细胞凋亡状况及形态变化；流式细胞仪检测足细胞凋亡率；Western Blot检测细胞内自噬标志蛋白SIRT1、P62及促凋亡蛋白BNIP3、Bax表达水平。[结果] 流式细胞仪检测结果显示通络益肾方中、低剂量组可降低高糖诱导的足细胞凋亡率（P<0.01或P<0.05），高剂量组不能改善凋亡情况（P>0.05）；Hoechst33342荧光染色观察结果也证实通络益肾方中、低剂量组可降低高糖诱导的足细胞凋亡率；蛋白印迹结果显示，与高糖组相比，通络益肾方中、低剂量组自噬标志蛋白SIRT1表达升高，自噬标志蛋白P62及促凋亡蛋白BNIP3、Bax蛋白表达下降（P<0.05或P<0.01），高剂量组SIRT1、BNIP3、P62、Bax蛋白表达未见明显改变（P>0.05）。[结论] 中剂量、低剂量通络益肾方能够启动细胞自噬减少高糖刺激下体外培养足细胞凋亡，降低细胞凋亡率及凋亡蛋白的表达。

Abstract:

[Objective] Based on the treatment method of "tonifying kidney and collaterals,tonifying spleen and kidney",to investigate the regulatory effect of Tongluo Yishen Decoction on the expressions of autophagy and apoptosis proteins SIRT1,BNIP3,P62 and bcl-2 in mouse glomerular podocytes cultured in vitro with high glucose. [Methods] Forty mature SPF-grade SD male rats were randomly divided into the normal group,the large,medium and small dose groups of traditional Chinese medicine,10 rats in each group. According to the method of human and rat body surface area conversion,the concentration of traditional Chinese medicine required by rats was calculated:large dose concentration (4.76 g/mL),medium dose concentration (2.38 g/mL),and small dose concentration (1.19 g/mL). After 10 days of gavage,the drug-containing serum was taken for later use.Podocytes were divided into 6 groups:normal group (5.6 mmol/L glucose+10% normal rat serum),high glucose group (30 mmol/L glucose+10% normal rat serum),Tongluo Yishen Decoction large/medium/small dose intervention groups (30 mmol/L glucose+10% Tongluo Yishen Decoction large/medium/small dose rat serum) and hypertonic group (24.5 mmol/L mannitol+10% normal rat serum). The cells were cultured for 48 h according to the requirements of each group and collected. The apoptosis status and morphological changes of podocytes in each group were observed after fluorescence staining with Hoechst 33342. The apoptosis rate of podocytes was measured by flow cytometry. Expression levels of SIRT1,BNIP3,P62 and Bax proteins in cells were detected by Western Blot. [Results] The results of flow cytometry showed that the medium and small dose of Tongluo Yishen Decoction could reduce the apoptosis rate of podocytes induced by high glucose (P<0.01 or P<0.05) and the high dose group could not improve apoptosis (P>0.05). The observation results of Hoechst33342 fluorescence staining also confirmed that the medium and small dose groups of Tongluo Yishen Decoction could reduce the apoptosis rate of podocytes induced by high glucose. Western blot results showed that compared with the high-glucose group,SIRT1 protein expression was increased in the medium and small dose groups of tongluo yishen prescription,while BNIP3,P62 and Bax protein expression was decreased (P<0.05 or P<0.01). Protein expressions of SIRT1,BNIP3,P62 and Bax were not significantly changed in the high-dose group (P>0.05). [Conclusion] Medium and small doses of Tongluo Yishen Decoction can activate autophagy to reduce apoptosis of podocytes in vitro under the stimulation of high glucose,and reduce apoptosis rate and expression of apoptotic protein.