黄连大黄药对调控ROS/NF-κB/GSDMD信号通路对2型糖尿病大鼠胰岛β细胞焦亡的影响

Effects of Huanglian(Coptidis Rhizoma)and Dahuang(Rhei Radix et Rhizoma)on Pyroptosis of Pancreaticβ-cells in Type 2 Diabetic Rats by Regulation of ROS/NF-κB/GSDMD Signaling Pathway

目的基于活性氧(reactive oxygen species,ROS)/核转录因子κB(nuclear transcription factor-κB,NF-κB)/消皮素D(gasdermin D,GSDMD)焦亡信号通路探讨黄连大黄药对发挥2型糖尿病(type 2 diabetes mellitus,T2DM)胰岛β细胞保护作用的分子机制。方法选取自发性T2DM大鼠—Goto-Kakizaki(GK)大鼠构建动物模型,24只符合成模标准的GK大鼠随机分为模型组、二甲双胍组(0.1 g·kg^(-1)·d^(-1))、黄连大黄高剂量组(4.72 g·kg^(-1)·d^(-1))及低剂量组(2.36 g·kg^(-1)·d^(-1)),并随机选取6只正常Wistar大鼠设为正常组,连续灌胃干预8周后采用免疫荧光法检测大鼠胰腺组织胰岛素(insulin,INS)阳性表达,RT-PCR法检测细胞增殖相关细胞周期蛋白D1(cyclin D1,CCND1)、髓细胞瘤病毒癌基因(cellular-myelocytomatosis viral oncogene,c-Myc)mRNA表达,TUNEL染色检测胰岛细胞死亡,流式细胞术检测ROS水平,Western blot法检测NF-κB p65、GSDMD及消皮素D N端片段(gasdermin D-N,GSDMD-N)蛋白的表达,ELISA法检测白介素1β(interleukin 1β,IL^(-1)β)水平。结果与正常组比较,模型组大鼠胰腺组织INS平均光密度值显著降低,CCND1、c-Myc mRNA表达显著下调,TUNEL阳性率、ROS水平和NF-κB p65、GSDMD-N蛋白表达以及IL-1β水平均显著升高(P均<0.01);与模型组比较,黄连大黄低剂量组INS平均光密度值有效升高,CCND1、c-Myc mRNA表达有效上调,TUNEL阳性率、ROS水平以及NF-κB p65、GSDMD-N蛋白表达、IL-1β水平均明显降低(P<0.01,P<0.05),黄连大黄高剂量组仅c-Myc mRNA表达有效上调,NF-κB p65蛋白表达明显降低(P均<0.05)。结论黄连大黄药对可能通过ROS/NF-κB/GSDMD信号通路抑制细胞焦亡,促进增殖进而起到保护T2DM大鼠胰岛β细胞的作用。

Objective To explore the molecular mechanism of the protective effect of Huanglian(Coptidis Rhizoma)and Dahuang(Rhei Radix et Rhizoma)on exerting pancreaticβ-cells protection in type 2 diabetes mellitus(T2DM)based on the reactive oxygen species(ROS)/nuclear transcription factor-κB(NF-κB)/gasdermin D(GSDMD)pyroptosis signaling path-way.Methods Spontaneous T2DM rats[Goto-Kakizaki(GK)rats]were selected to construct the animal models,and 24 GK rats that met the modeling criteria were randomly divided into the model group,the metformin group(0.1 g·kg^(-1)·d^(-1)),the Huan-glian(Coptidis Rhizoma)-Dahuang(Rhei Radix et Rhizoma)high-dose(4.72 g·kg^(-1)·d^(-1))and low-dose(2.36 g·kg^(-1)·d^(-1))groups.Moreover,6 normal Wistar rats were randomly selected and set as the normal group.After 8 weeks of continuous gavage intervention,the positive expression of insulin(INS)in rat pancreatic tissue was detected by immunofluorescence.Real-time PCR was used to detect the expressions of cell proliferation-associated cell cycle protein D1(CCND1)and my-elomavirus oncogene(c-Myc)mRNA.TUNEL staining was used to detect cell death.Flow cytometry measured ROS levels,and the expressions of NF-κB p65,GSDMD and gasdermin D N-terminal fragment(GSDMD-N)proteins were detected by Western blot.ELISA was used to detect interleukin 1β(IL^(-1)β)levels.Results Compared with those of the normal group,the aver-age optical density of pancreatic tissue INS was significantly lower,the expressions of CCND1 and c-Myc mRNA were significantly down-regulated,and the TUNEL positivity rate,ROS level,NF-κB p65,GSDMD-N protein expression and IL-1βlevel were significantly higher in the model group(all P<0.01).Compared with those of the model group,the average optical density value of INS in the Huanglian(Coptidis Rhizoma)-Dahuang(Rhei Radix et Rhizoma)lowdose group was effectively increased,the expressions of CCND1 and c-Myc mRNA were effectively up-regulated,and the TUNEL positive rate,ROS level,NF-κB p65 and GSDMD-N protein expression and IL-1βlevel were significantly reduced(P<0.01,P<0.05),while only c-Myc mRNA expression was effectively up-regulated and NF-κB p65 protein expression was significantly reduced in the Huanglian(Coptidis Rhizoma)-Dahuang(Rhei Radix et Rhizoma)high-dose group(P<0.05 for both).Conclusion Huanglian(Coptidis Rhizoma)and Dahuang(Rhei Radix et Rhizoma)may inhibit cell pyroptosis through the ROS/NF-κB/GSDMD signaling pathway and promote proliferation,thereby protecting the pancreaticβ-cells of T2DM rats.