基于TXNIP/GSDMD通路探讨补阳还五汤抑制高血压病大鼠内皮焦亡作用机制

Investigation of the Mechanism of Buyang Huanwu Decoction(补阳还五汤)in Inhibiting Endothelial Pyrodeath in Hypertensive Rats Based on TXNIP/GSDMD Pathway

目的通过观察补阳还五汤对高血压病大鼠硫氧还蛋白互作蛋白-消皮素D(TXNIP/GSDMD)信号转导通路的影响,从焦亡角度探讨补阳还五汤对高血压病大鼠主动脉血管内皮损伤的保护机制。方法将大鼠随机分为空白对照组,模型组,补阳还五汤低、中、高剂量组,氢氯噻嗪组,每组10只,所有大鼠于SPF级动物实验中心普通饲料适应性饲养7 d。空白对照组每日给予低盐饲料喂养,其余各组采用高盐饲料喂养,同时,补阳还五汤低、中、高剂量组分别以0.275、0.55、1.1 g/mL浓度中药干预,氢氯噻嗪组以10 mg/(kg·d)浓度药物干预。3周后,观察到大鼠血压比造模前升高大于或等于30 mm Hg(1 mm Hg≈0.133 kPa),且收缩压(SBP)≥160 mm Hg即高血压病大鼠造模成功。取大鼠主动脉进行检测,HE染色观察大鼠主动脉病理形态改变;采用ELISA检测大鼠主动脉中血管紧张素Ⅱ(AngⅡ)、白细胞介素18(IL-18)、白细胞介素1β(IL-1β)、超氧化物歧化酶(superoxide dismutase,SOD)以及丙二醛(malondialdehyde,MDA)蛋白表达;荧光定量PCR检测大鼠主动脉中TXNIP、GSDMD、还原性辅酶Ⅱ氧化酶4(NOX4)、IL-1β的mRNA表达,免疫印迹实验(Western blot)检测大鼠主动脉中GSDMD的蛋白表达,免疫组化(IHC)检测大鼠主动脉中TXNIP、NOD样受体蛋白3(NLRP3)、GSDMD的蛋白表达。结果与空白对照组相比,模型组大鼠血压明显升高,主动脉内外膜都有一定脱落,界线模糊,血管中膜弹性纤维结构比较疏松,排列有一定紊乱;TXNIP、GSDMD、IL-1β、NOX4的mRNA表达上升(P<0.05);TXNIP、NLRP3、GSDMD、IL-1β、IL-18、AngⅡ、MDA的蛋白表达升高(P<0.05),SOD蛋白表达降低(P<0.05)。与模型组相比,补阳还五汤中剂量组和氢氯噻嗪组大鼠血压明显降低,血管内外膜更加完整、光滑,结构更加致密,界线较为清晰明显,补阳还五汤低、高剂量组内外膜仍有一定脱落,弹性纤维排列比较杂乱,变化不明显;药物干预后各组TXNIP、GSDMD、IL-1β、NOX4的mRNA表达均下降(P<0.05);补阳还五汤中剂量组和氢氯噻嗪组TXNIP、NLRP3、GSDMD、IL-1β、IL-18、AngⅡ、MDA的蛋白表达降低(P<0.05),SOD蛋白表达升高(P<0.05)。结论补阳还五汤可通过抑制氧化应激,调节TXNIP/GSDMD信号通路的分子表达,调控血管内皮焦亡,从而发挥保护血管、治疗高血压病的作用。

Objective To investigate the protective mechanism of Buyang Huanwu Decoction(补阳还五汤)on aortic endothelial injury in hypertensive rats from the perspective of pyroptosis-by observing the effect of Buyang Huanwu Decoction on thioredoxin interacting protein-descaline D(TXNIP/GSDMD)signal transduction pathway.Methods The rats were randomly divided into blank control group,model group,Buyang Huanwu Decoction low-dose,medium-dose and high-dose groups,and hydrochlorothiazide group,with 10 rats in each group.All the rats were fed with ordinary feed in SPF animal experimental center for 7 days.The control group was fed with low-salt diet every day,and the other groups were fed with high-salt diet.Meanwhile,the low-dose,medium-dose and high-dose groups were treated with 0.275,0.55 and 1.1 g/mL concentration of traditional Chinese medicine,respectively,and the hydrochlorothiazide group was treated with 10 mg/(kg·d)concentration of drugs.After 3 weeks,it was observed that the blood pressure of the rats was greater than or equal to 30 mm Hg(1 mm Hg≈0.133 kPa)higher than that before modeling,and the systolic blood pressure(SBP)was≥160 mm Hg,indicating that the modeling of hypertensive rats was successful.The aorta of rats was detected and the pathological morphology of aorta was observed by HE staining.The expression of angiotensin Ⅱ(AngⅡ),interleukin 18(IL-18),interleukin 1β(IL-1β),superoxide dismutase(SOD)and malondialdehyde(MDA)in aorta of rats was determined by ELISA.Fluorescence quantitative PCR was used to detect TXNIP,GSDMD,reduced Coenzyme Ⅱ oxidase 4,IL-1β mRNA expression in the aorta of rats.Western blot assay was used to detect the protein expression of GSDMD in the aorta of rats.The expression of TXNIP,NLRP3 and GSDMD in rat aorta was detected by immunohistochemistry(IHC).Results Compared with the blank control group,the blood pressure of the model group was significantly increased,the inner and outer aortic membranes were somewhat detached,the boundaries were blurred,the elastic fiber structure of the vascular media was loose,and the arrangement was somewhat disorganized.The mRNA contents of TXNIP,GSDMD,IL-1β and NOX4 were increased(P<0.05).The expression levels of TXNIP,NLRP3,GSDMD,IL-1β,IL-18,AngⅡ and MDA of all medicine groups were increased(P<0.05),while the expression levels of SOD were decreased(P<0.05).Compared with model group,the blood pressure of Buyang Huanwu Decoction medium-dose group and hydrochlorothiazide group were significantly reduced,and the inner and outer membranes of blood vessels were more complete and smooth,the structure was more dense,and the boundaries were more clear and obvious.The inner and outer membranes of Buyang Huanwu Decoction lowdose and high-dose groups still had some shedding,and the elastic fibers were more disorderly,and the change was not obvious.The mRNA contents of TXNIP,GSDMD,IL-1β and NOX4 were all decreased after drug intervention(P<0.05).The expression of TXNIP,NLRP3,GSDMD,IL-1β,IL-18,AngⅡ and MDA protein was decreased(P<0.05)and the expression of SOD protein was increased(P<0.05)in medium dose group and hydrochlorothiazide group.Conclusion Buyang Huanwu Decoction can inhibit oxidative stress,regulate the molecular expression of TXNIP/GSDMD signaling pathway,regulate vascular endothelial pyrosis,and thus play a role in protecting blood vessels and treating hypertension.