目的 探究黄芪甲苷通过调控环磷酸腺苷(cyclic adenosine monophosphate，cAMP)/蛋白激酶A(protein kinase A，PKA)信号通路抑制铁死亡减轻单侧输尿管梗阻(unilateral ureteral obstruction，UUO)大鼠肾纤维化的相关机制。 方法 选用SPF级健康雄性SD大鼠建立UUO模型，随机分为4组(假手术组、模型组、黄芪甲苷组、氯沙坦钾组，每组6只大鼠)，手术后1天开始灌胃，连续14天。检测血清肌酐(Scr)、尿素氮(BUN)、cAMP水平，苏木精伊红(HE)染色及马松染色观察梗阻侧肾脏组织病理改变，免疫组化法检测肾组织纤维化指标[α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤维连接蛋白(fibronectin,FN)、I型胶原蛋白(collagen type I,COL-I)]，Western blotting法检测PKA及铁死亡相关指标[谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、血红素加氧酶-1(heme oxygenase 1,HO-1)、溶质载体家族7成员11(solute carrier family 7 member 11,xCT)]的表达。 结果 假手术组、模型组、黄芪甲苷组、氯沙坦钾组的Scr比较无统计学差异(F=2.815、P=0.065)。较假手术组，模型组的BUN升高(t=26.306，P＜0.001)；较模型组，黄芪甲苷组、氯沙坦钾组的BUN下降(t=-5.241、 -3.469，P＜0.001、P=0.002)。HE及马松染色结果显示模型组大鼠梗阻侧肾脏明显纤维化，黄芪甲苷组与氯沙坦钾组肾脏纤维化程度均较模型组轻。模型组较假手术组的α-SMA、FN、COL-I(t=17.728、9.202、13.710，均P＜0.001)、cAMP(t=9.601，P＜0.001)、PKA(t=32.321，P＜0.001)表达升高；黄芪甲苷组、氯沙坦钾组的α-SMA(t=-11.457、-5.519，P＜0.001、P=0.001)、FN(t=-6.301、-4.725，P＜0.001、 P=0.001)、COL-I(t=-6.087、-3.243，P＜0.001、P=0.012)、cAMP(t=-6.629、-5.809，均P＜0.001)、PKA(t=-22.754、-23.294，均P＜0.001)表达较模型组均降低；模型组的GPX4、HO-1、xCT较假手术组降低(t= -38.397、-41.713、-56.779，均P＜0.001)，黄芪甲苷组、氯沙坦钾组的GPX4(t=25.504、16.786，均P＜0.001)、HO-1(t=10.611、42.007，均P＜0.001)、xCT(t=7.192、3.181，P＜0.001、=0.013)表达较模型组均升高。 结论 黄芪甲苷可改善UUO大鼠肾功能，缓解肾纤维化，部分机制可能是通过调控cAMP/PKA信号通路抑制铁死亡来抑制肾纤维化。
Objective To investigate the mechanism by which astragaloside IV alleviates renal fibrosis in unilateral ureteral obstruction (UUO) rats by inhibiting ferroptosis through modulation of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway. Methods Specific pathogen-free (SPF) male Sprague-Dawley (SD) rats were used to establish UUO models and randomly divided into four groups (n=6 per group): sham operation group, model group, AS-IV group [40 mg/(kg·d)], and losartan group [10.3 mg/(kg·d)]. Intragastric administration began one day post-surgery and continued for 14 days. Serum creatinine (Scr), blood urea nitrogen (BUN), and cAMP levels were measured. Renal histopathology was assessed via hematoxylin-eosin (HE) and Masson staining. Immunohistochemistry evaluated fibrosis markers [α-smooth muscle actin (α-SMA), fibronectin (FN), collagen type I (COL-I)], while Western blotting analyzed PKA and ferroptosis-related markers [glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), solute carrier family 7 member 11 (xCT)]. Results No significant differences in Scr were observed among groups (F=2.815, P=0.065). Compared to the sham group, the model group exhibited elevated BUN (t=26.306, P＜0.001), which was reduced in the astragaloside IV and losartan potassium groups (t=−5.241, −3.469; P＜0.001, P=0.002, respectively). HE and Masson staining revealed severe renal fibrosis in the obstructed kidneys of rats in the model group, while fibrosis was attenuated in the astragaloside IV and losartan potassium groups. Compared with the sham operation group, the model group exhibited increased expression of α-SMA, FN, COL-I (t =17.728, 9.202, 13.710, all P＜0.001), cAMP levels (t =9.601, P＜0.001), and PKA (t =32.321, P＜0.001). In the astragaloside IV and losartan potassium groups, the expression of α-SMA (t =-11.457, -5.519, P＜0.001, P =0.001), FN (t =-6.301, -4.725, P＜0.001, P =0.001), COL-I (t =-6.087, -3.243, P＜0.001, P=0.012), cAMP levels (t =-6.629,-5.809, both P＜0.001), and PKA (t =-22.754, -23.294, both P＜0.001) was decreased compared with the model group. Compared with the sham operation group, the model group showed decreased expression of GPX4, HO-1, and xCT (t =-38.397, -41.713, -56.779, all P＜0.001). In the astragaloside IV and losartan potassium groups, the expression of GPX4 (t =25.504, 16.786, both P＜0.001), HO-1 (t=10.611, 42.007, both P＜0.001), and xCT (t=7.192, 3.181, P＜0.001, P =0.013) was increased compared with the model group. Conclusions Astragaloside IV improves renal function and mitigates fibrosis in UUO rats, potentially by suppressing ferroptosis through regulation of the cAMP/PKA signaling pathway.
[1] Francis A, Harhay M N, Ong A, et al. Chronic kidney disease and the global public health agenda: an international consensus[J]. Nature Reviews Nephrology, 2024: 1-13.
[2] Zhang L, Wang F, Wang L, et al. Prevalence of chronic kidney disease in China: a cross-sectional survey[J]. The lancet, 2012, 379(9818): 815-822.
[3] Ruiz-Ortega M, Rayego-Mateos S, Lamas S, et al. Targeting the progression of chronic kidney disease[J]. Nature Reviews Nephrology, 2020, 16(5): 269-288.
[4] 赖玮婧,黄蓉双,王波等.肾脏病中铁死亡的病理生理机制和药物疗法研究进展[J].中华肾脏病杂志,2024,40(5):411-417.
[5] Li D, Liu Y, Zhan Q, et al. Astragaloside IV Blunts Epithelial–Mesenchymal Transition and G2/M Arrest to Alleviate Renal Fibrosis via Regulating ALDH2-Mediated Autophagy[J]. Cells, 2023, 12(13): 1777.
[6] Zhou X, Sun X, Gong X, et al. Astragaloside IV from Astragalus membranaceus ameliorates renal interstitial fibrosis by inhibiting inflammation via TLR4/NF-кB in vivo and in vitro[J]. International Immunopharmacology, 2017, 42: 18-24.
[7] Wang X, Gao Y, Tian N, et al. Astragaloside IV improves renal function and fibrosis via inhibition of miR-21-induced podocyte dedifferentiation and mesangial cell activation in diabetic mice[J]. Drug design, development and therapy, 2018: 2431-2442.
[8] Weng L, Wang W, Su X, et al. The effect of cAMP-PKA activation on TGF-β1-induced profibrotic signaling[J]. Cellular Physiology and Biochemistry, 2015, 36(5): 1911-1927.
[9] Delrue C, Speeckaert R, Moresco R N, et al. Cyclic Adenosine Monophosphate Signaling in Chronic Kidney Disease: Molecular Targets and Therapeutic Potentials[J]. International Journal of Molecular Sciences, 2024, 25(17): 9441.
[10] 黄倩,张素萍,施子禄.人参多糖通过cAMP/PKA/CREB信号通路抗糖尿病肾病肾纤维化作用机制研究[J].中国药理学通报,2018,34(05):695-701.
[11] Guan Q, Wang Z, Hu K, et al. Melatonin ameliorates hepatic ferroptosis in NAFLD by inhibiting ER stress via the MT2/cAMP/PKA/IRE1 signaling pathway[J]. International journal of biological sciences, 2023, 19(12): 3937.
[12] Huang R, Fu P, Ma L. Kidney fibrosis: from mechanisms to therapeutic medicines[J]. Signal Transduction and Targeted Therapy, 2023, 8(1): 129.
[13] Li L, He M, Tang X, et al. Proteomic landscape of the extracellular matrix in the fibrotic kidney[J]. Kidney International, 2023, 103(6): 1063-1076.
[14] Song Y, Liu W, Tang K, et al. Mangiferin Alleviates Renal Interstitial Fibrosis in Streptozotocin‐Induced Diabetic Mice through Regulating the PTEN/PI3K/Akt Signaling Pathway[J]. Journal of Diabetes Research, 2020, 2020(1): 9481720.
[15] Dixon S J, Lemberg K M, Lamprecht M R, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death[J]. cell, 2012, 149(5): 1060-1072.
[16] Dodson M, Castro-Portuguez R, Zhang D D. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis[J]. Redox biology, 2019, 23: 101107.
[17] Lane D J R, Metselaar B, Greenough M, et al. Ferroptosis and NRF2: an emerging battlefield in the neurodegeneration of Alzheimer's disease[J]. Essays in Biochemistry, 2021, 65(7): 925-940.
[18] Adedoyin O, Boddu R, Traylor A, et al. Heme oxygenase-1 mitigates ferroptosis in renal proximal tubule cells[J]. American Journal of Physiology-Renal Physiology, 2018, 314(5): F702-F714.
[19] Kwon M Y, Park E, Lee S J, et al. Heme oxygenase-1 accelerates erastin-induced ferroptotic cell death[J]. Oncotarget, 2015, 6(27): 24393.
[20] Tu H, Tang L J, Luo X J, et al. Insights into the novel function of system Xc-in regulated cell death[J]. European Review for Medical & Pharmacological Sciences, 2021, 25(3).
[21] Seibt T M, Proneth B, Conrad M. Role of GPX4 in ferroptosis and its pharmacological implication[J]. Free Radical Biology and Medicine, 2019, 133: 144-152.
[22] Meng X, Nikolic-Paterson D J, Lan H Y. TGF-β: the master regulator of fibrosis[J]. Nature Reviews Nephrology, 2016, 12(6): 325-338.
[23] Delrue C, Speeckaert R, Moresco R N, et al. Cyclic Adenosine Monophosphate Signaling in Chronic Kidney Disease: Molecular Targets and Therapeutic Potentials[J]. International Journal of Molecular Sciences, 2024, 25(17): 9441.
[24] Deb D K, Bao R, Li Y C. Critical role of the cAMP-PKA pathway in hyperglycemia-induced epigenetic activation of fibrogenic program in the kidney[J]. The FASEB Journal, 2017, 31(5): 2065.
[25] Zhang L, Liu W, Li S, et al. Astragaloside IV alleviates renal fibrosis by inhibiting renal tubular epithelial cell pyroptosis induced by urotensin II through regulating the cAMP/PKA signaling pathway[J]. Plos one, 2024, 19(5): e0304365.
