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目的：探讨肾消通络方调控过氧化物酶体增殖物激活受体-γ共激活因子-1α（PGC-1α）/沉默调节蛋白3(SIRT3)–缺氧诱导因子-1α（HIF-1α）/丙酮酸激酶M2型(PKM2)信号通路，对高糖诱导HK-2细胞损伤的影响。方法：将HK-2细胞分为正常糖对照组（5.5 mmol/L葡萄糖）、高糖对照组（30 mmol/L葡萄糖）、高糖+肾消通络方10%含药血清组、高糖+siSIRT3组、高糖+siSIRT3+肾消通络方10%含药血清组及正常糖+siSIRT3组。采用SweTransRNA进行siSIRT3瞬时转染，另设阴性对照正常糖+siNC组与高糖+siNC组。采用CCK-8法检测细胞活力并筛选最佳含药血清浓度；流式细胞术JC-1检测线粒体膜电位；荧光探针法检测ROS；实时荧光定量聚合酶链式反应（Real-time PCR）检测SIRT3、PGC-1α、HIF-1α、PKM2、FIS1、DRP1、MFN2、OPA1 mRNA表达。蛋白免疫印迹法（Western blot）检测SIRT3、PGC-1α、HIF-1α、PKM2、FIS1、DRP1、MFN2、OPA1蛋白表达。结果：15.31 g/kg肾消通络方含药血清浓度筛选提示10%组细胞活力最高；敲低SIRT3后细胞活力在正常糖与高糖条件下均降低（P<0.05），转染阴性对照（siNC）组在正常糖与高糖条件下与相应未转染组接近；与正常糖对照组比较，高糖对照组线粒体膜电位去极化、ROS水平及HIF-1α、PKM2、FIS1、DRP1 mRNA与蛋白表达均升高（P<0.05），PGC-1α、SIRT3、MFN2、OPA1 mRNA与蛋白表达均降低（P<0.05）；加入含药血清后，线粒体膜电位去极化、ROS水平、HIF-1α、PKM2、FIS1、DRP1 mRNA及蛋白表达均下调（P<0.05），PGC-1α、SIRT3、MFN2、OPA1 mRNA与蛋白表达上调；在正常糖和高糖条件下敲低SIRT3后，线粒体膜电位去极化、ROS水平、HIF-1α、PKM2、FIS1、DRP1 mRNA与蛋白表达均升高（P<0.05），PGC-1α、SIRT3、MFN2、OPA1 mRNA与蛋白表达降低；在高糖+siSIRT3基础上加入含药血清后上述趋势得到纠正（P<0.05），但SIRT3 mRNA与蛋白表达改变无统计学意义；转染阴性对照（siNC）组在正常糖与高糖条件下各基因表达与相应未转染组mRNA表达水平相比无明显改变。结论：肾消通络方可能通过调控PGC-1α/SIRT3–HIF-1α/PKM2信号通路改善代谢重编程及线粒体动力学，从而减轻高糖诱导的细胞损伤。
Abstract：Objective: To investigate the effects of Shenxiao Tongluo (肾消通络) Formula on high glucose-induced injury in HK-2 cells through regulation of the peroxisome proliferation-activated receptor gamma coactivator-1α (PGC-1α)/sirtuin 3 (SIRT3)–hypoxia-inducible factor-1α (HIF-1α)/pyruvate kinase M2 (PKM2) signaling pathway. Methods: HK-2 cells were allocated into a normal glucose (5.5 mmol/L) group， a high glucose (30 mmol/L ) group， a high glucose + Shenxiao Tongluo Formula-containing serum (10%) group， a high glucose + siSIRT3 group， a high glucose + siSIRT3 + Shenxiao Tongluo Formula-containing serum (10%) group， and a normal glucose + siSIRT3 group. Transient transfection with siSIRT3 was performed through SweTransRNA. Negative control groups—normal glucose + siNC and high glucose + siNC groups—were also established. Cell viability was measured by the CCK-8 assay to determine the optimal concentration of Shenxiao Tongluo Formula-containing serum. Mitochondrial membrane potential was detected by JC-1 staining with flow cytometry. Reactive oxygen species (ROS) levels were measured through a fluorescent probe assay. The mRNA levels of SIRT3， PGC1A， HIF1A， PKM， fission 1 (FIS1)， dynamin-related protein 1 (DNM1L)， mitofusin 2 (MFN2)， and optic atrophy 1 (OPA1) were determined by real-time PCR. The protein levels of SIRT3， PGC-1α， HIF-1α， PKM2， FIS1， DRP1， MFN2， and OPA1 were determined by Western blot. Results: Concentration screening of 15.31 g/kg Shenxiao Tongluo Formula-containing serum showed that cell viability was highest in the 10% group. After SIRT3 knockdown， cell viability was decreased under both normal glucose and high glucose conditions (P<0.05). In the negative control transfection (siNC) groups， cell viability under normal glucose and high glucose conditions was similar to that in the corresponding non-transfected groups. Compared with the normal glucose group， the high glucose group showed increased mitochondrial membrane potential depolarization， an elevated ROS level， upregulated protein and mRNA levels of HIF-1α， PKM2， FIS1， and DRP1 (P<0.05)， and downregulated protein and mRNA levels of PGC-1α， SIRT3， MFN2， and OPA1 were decreased (P<0.05). The treatment with Shenxiao Tongluo Formula-containing serum decreased the mitochondrial membrane potential depolarization， lowered the ROS level， downregulated the protein and mRNA levels of HIF-1α， PKM2， FIS1， and DRP1 (P<0.05)， and upregulated the protein and mRNA levels of PGC-1α， SIRT3， MFN2， and OPA1. Under both normal glucose and high glucose conditions， SIRT3 knockdown increased mitochondrial membrane potential depolarization， raised the ROS level， upregulated the protein and mRNA levels of HIF-1α， PKM2， FIS1， and DRP1 (P<0.05)， and downregulated the mRNA and protein levels of PGC-1α， SIRT3， MFN2， and OPA1. On the basis of the high glucose + siSIRT3 group， addition of Shenxiao Tongluo Formula-containing serum reversed the above changes (P<0.05)， while the changes in the protein and mRNA levels of SIRT3 were not statistically significant. In the negative control transfection (siNC) groups， no significant differences were observed in the mRNA levels of these genes under either normal glucose or high glucose conditions compared with the corresponding non-transfected groups. Conclusion: Shenxiao Tongluo Formula can regulate the PGC-1α/SIRT3–HIF-1α/PKM2 signaling pathway to ameliorate metabolic reprogramming and improve mitochondrial dynamics， thereby alleviating high glucose-induced cellular injury.
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基本信息:
DOI：10.13412/j.cnki.zyyl.20261022.001
中图分类号:R285.5
引用信息:
[1]焦文浩,赵冬,宋天毅,等.肾消通络方调控PGC-1α/SIRT3–HIF-1α/PKM2信号通路缓解高糖诱导的HK-2细胞损伤[J].中药药理与临床().DOI:10.13412/j.cnki.zyyl.20261022.001.
基金信息:
河北省中医药联合基金重点项目（编号：H2023423008）
2026-10-23
2026-10-23
2026-10-23