参芪泄浊饮通过调控ERK1/2-mTOR信号通路抑制过度自噬延缓大鼠肾纤维化

doi:10.12122/j.issn.1673-4254.2026.10.03

摘要/Abstract

摘要：

目的探讨参芪泄浊饮（SQXZD）延缓肾纤维化的作用机制。方法采用网络药理学富集SQXZD延缓肾纤维化的靶点及信号通路，分子对接分析SQXZD成分与相关靶点的结合能力。体内实验：将49只SPF级雄性SD大鼠随机分为空白组，假手术组，模型组，氯沙坦组（4.6 mg·kg^-1·d^-1）及SQXZD低、中、高剂量（9.7、19.4、38.8 g·kg^-1·d^-1）组，7只/组。除空白组及假手术组，其他组大鼠均以单侧输尿管结扎法复制肾纤维化模型。造模24 h后开始灌胃，14 d后取材，Masson染色观察肾组织胶原分布，并检测纤维化相关指标（E-Cadherin）、自噬相关指标（LC3B、p62/SQSTM1）及ERK1/2-mTOR信号通路表达。体外实验：将大鼠肾正常成纤维细胞（NRK-49F）及肾小管上皮细胞（NRK-52E）各分为对照组（未处理）、模型组（TGF-β₁处理）、空白血清组（TGF-β₁+空白血清处理）、二甲基亚砜（DMSO）组（TGF-β₁+DMSO处理）、含药血清组（TGF-β₁+SQXZD含药血清处理）、间双没食子酸（DGA）组（TGF-β₁+DGA处理）、含药血清+U0126组（TGF-β₁+SQXZD含药血清+U0126处理）、DGA+U0126组（TGF-β₁+DGA+U0126处理），免疫荧光确定纤维化诱导条件，CCK-8法确定SQXZD含药血清及DGA处理条件，丹酰尸胺染色及透射电子显微镜观察各组细胞自噬水平，检测纤维化相关指标（α-SMA、Col-Ⅰ、E-Cadherin）、自噬相关指标（LC3B、p62/SQSTM1）及ERK1/2-mTOR信号通路表达。结果 SQXZD主要活性成分为4-O-甲基芍药苷、DGA等54个，与IGF1、KDR、ALB等靶点结合紧密。体内实验显示，与假手术组比较，模型组大鼠术侧肾组织LC3B-Ⅱ表达上调，E-cadherin、p62/SQSTM1、p-mTOR/mTOR表达下调（P<0.01）。与模型组比较，各给药组大鼠术侧肾组织LC3B-Ⅱ表达下调，E-cadherin、p62/SQSTM1、p-mTOR/mTOR表达上调（P<0.05）。体外实验显示，与对照组比较，10 ng/mL TGF-β₁处理NRK-49F细胞72 h与5 ng/mL TGF-β₁处理NRK-52E细胞36 h后α-SMA表达上调（P<0.01）。与模型组比较，15%体积SQXZD含药血清分别处理TGF-β₁诱导的NRK-49F细胞24 h、NRK-52E细胞12 h可抑制细胞活力（P<0.05）；NRK-49F及NRK-52E细胞DGAIC₅₀分别为90.15、610.66 ng/mL。与对照组比较，模型组细胞自噬荧光增强、自噬体数量增多，α-SMA、Col-Ⅰ（NRK-49F）、LC3B-Ⅱ、p-ERK1/2/ERK1/2表达上调，E-cadherin（NRK-52E）、p62/SQSTM1、p-mTOR/mTOR表达下调（P<0.01）。与模型组比较，含药血清组、含药血清+U0126组、DGA组、DGA+U0126组细胞自噬荧光减弱、自噬体数量减少，α-SMA、Col-Ⅰ（NRK-49F）、LC3B-Ⅱ、p-ERK1/2/ERK1/2表达下调，E-cadherin（NRK-52E）、p62/SQSTM1、p-mTOR/mTOR表达上调（P<0.05）。结论 SQXZD可抑制成纤维细胞活化及上皮-间质转化以延缓肾纤维化，其机制可能是通过调控ERK1/2-mTOR信号通路以抑制过度自噬。

关键词: 参芪泄浊饮, 肾纤维化, 成纤维细胞活化, 上皮-间质转化, 自噬, ERK1/2-mTOR信号通路, 间双没食子酸

Abstract:

Objective To investigate the mechanism of Shenqi Xiezhuo Decoction (SQXZD) for delaying progression of renal fibrosis (RF). Methods Network pharmacology and molecular docking were used to identify the active components of SQXZD and their targets and pathways. Forty-nine male SD rats were randomly divided into control group, sham-operated group, unilateral ureteral obstruction (UUO)-induced RF model group, losartan (4.6 mg/kg) group, and low-, medium-, high-dose SQXZD (9.7, 19.4, and 38.8 g/kg, respectively) groups. Renal tissues of the rats were collected to examine collagen distribution with Masson staining and detect expressions of E-cadherin, LC3B, p62/SQSTM1, and the ERK1/2-mTOR pathway. In TGF-β₁-induced NRK-49F and NRK-52E cells, the effects of blank rat serum, DMSO, SQXZD-medicated serum, digallic acid (DGA), SQXZD-medicated serum combined with U0126, or DGA combined with U0126 were assessed on autophagy levels and expressions of α‑SMA, collagen-I, E-eadherin, LC3B, p62/SQSTM1, and the ERK1/2-mTOR pathway. Results The 54 active components (including 4-O-methylpaeoniflorin and DGA) identified in SQXZD showed strong binding affinity to their targets including IGF1, KDR, and ALB. The RF rat models showed obviously increased renal expression of LC3B-II with lowered expressions of E-cadherin, p62/SQSTM1, and p-mTOR/mTOR, and these changes were significantly ameliorated by SQXZD treatment. TGF‑β₁ treatment significantly upregulated α‑SMA expression in NRK-49F cells and NRK-52E cells, whose viability was effectively inhibited by SQXZD-medicated serum. The IC₅₀ of DGA was 90.15 ng/mL and 610.66 ng/mL in NRK-49F and NRK-52E cells, respectively. The TGF-β₁-induced cells showed enhanced autophagy with increased autophagosome, upregulated α‑SMA, collagen-I, LC3B-II, and p-ERK1/2/ERK1/2 expressions, and downregulated E-cadherin, p62/SQSTM1, and p-mTOR/mTOR expressions. These changes were significantly reversed by treatment with SQXZD-medicated serum or DGA, either alone or in combination with U0126. Conclusion SQXZD inhibits fibroblast activation and epithelial-mesenchymal transition to ameliorate renal fibrosis potentially by inhibiting excessive autophagy via regulating the ERK1/2-mTOR pathway.

Key words: Shenqi Xiezhuo Decoction, renal fibrosis, fibroblast activation, epithelial-mesenchymal transition, autophagy, ERK1/2-mTOR signaling pathway, digallic acid

卢晓宇, 庞天霄, 刘烨, 何学红, 李志明. 参芪泄浊饮通过调控ERK1/2-mTOR信号通路抑制过度自噬延缓大鼠肾纤维化[J]. 南方医科大学学报, 2026, 46(10): 2309-2324.

Xiaoyu LU, Tianxiao PANG, Ye LIU, Xuehong HE, Zhiming LI. Shenqi Xiezhuo Decoction ameliorates renal fibrosis in rats by inhibiting excessive autophagy via modulating the ERK1/2-mTOR signaling pathway[J]. Journal of Southern Medical University, 2026, 46(10): 2309-2324.

图/表 17

图1 参芪泄浊饮延缓肾纤维化的作用靶点Venn图

Fig.1 Venn diagram of the action targets of SQXZD for delaying progression of renal fibrosis.

表1 参芪泄浊饮延缓肾纤维化的前10个关键成分及Degree值

Tab.1 Top 10 key components of SQXZD for delaying progression of renal fibrosis and their degree values

Components	Degree	Components	Degree
benzoyl paeoniflorin	201	Digallic acid	131
Diisooctyl phthalate	161	Physciondiglucoside	129
4-O-methylpaeoniflorin_qt	153	Acanthoside B	128
Procyanidin B-5,3'-O-gallate	136	Poricoic acid B	128
Poricoic acid A	135	Sennoside D_qt	125

图2 参芪泄浊饮延缓肾纤维化的关键靶点

Fig.2 Key targets of SQXZD for delaying progression of renal fibrosis (Color intensity and node area are based on degree values).

图3 参芪泄浊饮核心活性成分与核心靶点的分子对接模式

Fig.3 Molecular docking of the core active components of SQXZD with their core targets. A: DGA-IGF1. B: DGA-KDR. C: 4-O-methylpaeoniflorin-SRC. D: 4-O-methylpaeoniflorin-IGF1. E: 4-O-methylpaeoniflorin-ALB. F: 4-O-methylpaeoniflorin-KDR. G: 4-O-methylpaeoniflorin-CASP3. H: 4-O-methylpaeoniflorin-HSP90AA1. I: 4-O-methylpaeoniflorin-EGFR. DGA: Digallic acid.

图4 参芪泄浊饮对UUO大鼠术侧肾组织胶原沉积的影响

Fig.4 Effect of SQXZD on collagen deposition in the surgical-side kidney in UUO rats (Original magnification: ×400).

图6 参芪泄浊饮对UUO大鼠术侧肾组织EMT及自噬相关蛋白表达的影响

Fig.6 Effect of SQXZD on expression of EMT and autophagy-related proteins in the surgical-side kidney in UUO rats. **P<0.01 vs Sham group; *P<0.05, #P<0.01 vs Model group, n=3.

图7 参芪泄浊饮对UUO大鼠术侧肾组织p-mTOR、mTOR表达的影响

Fig.7 Effect of SQXZD on the expression of p-mTOR and mTOR in the surgical-side kidney in UUO rats. **P<0.01 vs Sham group; *P<0.05, #P<0.01 vs Model group, n=3.

图9 不同浓度TGF-β1处理后对NRK-52E细胞α-SMA表达的影响

Fig.9 Effects of different concentrations of TGF-β1 on α-SMA expression in NRK-52E cells (×400). Induction time: A, 12 h; B, 24 h; C, 36 h; D, 48 h. *P<0.05, #P<0.01 vs Control group; △P<0.01 vs 12 h-induced group by 10 ng/mL TGF-β1; ▲P<0.05 vs 12 h-induced group by 12.5 ng/mL TGF-β1 and 24 h-induced group by 10 ng/mL TGF-β1. n=3.

表2 不同体积的参芪泄浊饮含药血清对NRK-49F及NRK-52E细胞活力的影响

Tab.2 Effect of different Dilutions of SQXZD-medicated serum on viability of NRK-49F and NRK-52E cells (Mean±SD, n=3)

Item	Blank group	Control group	Dilutions of SQXZD-medicated serum groups (%)
Item	Blank group	Control group	2.5	5	7.5	10	12.5	15	17.5	20
NRK-49F	0.189±0.006	2.062±0.116	1.831±0.291	2.300±0.120	2.257±0.123	2.282±0.088	2.294±0.095	2.332±0.112*	2.164±0.180	2.117±0.087
NRK-52E	0.207±0.001	1.256±0.051	1.862±0.424	1.702±0.151	1.992±0.271	1.937±0.224	2.017±0.050*	2.284±0.146^#	2.238±0.153*	0.826±0.065

表3 参芪泄浊饮含药血清不同处理时间对TGF-β1诱导的NRK-49F及NRK-52E细胞活力的影响

Tab.3 Effect of different treatment durations with SQXZD-medicated serum on viability of TGF-β1-induced NRK-49F and NRK-52E cells (Mean±SD, n=3)

Group	Induction time NRK-49F		Induction time NRK-52E
Blank	1.711±0.053		1.830±0.018
Control	2.041±0.061		2.612±0.427
SQXZD-medicated serum	0 h	1.999±0.029	0 h	2.052±0.044
	24 h	2.014±0.104	12 h	2.093±0.039
	48 h	1.915±0.059*	24 h	2.049±0.038*

表4 不同浓度的DGA对NRK-49F及NRK-52E细胞活力的影响

Tab.4 Effect of different concentrations of DGA on viability of NRK-49F and NRK-52E cells (Mean±SD, n=3)

Cell	Blank group	Control group	Concentration of DGA (ng/mL)
Cell	Blank group	Control group	44.5	89	178	356	712
NRK-49F	1.927±0.046	1.808±0.015	2.259±0.150	0.980±0.080	0.273±0.009	0.611±0.019	0.921±0.016
NRK-52E	1.256±0.051	1.267±0.110	1.818±0.172	1.018±0.090	0.848±0.066	0.634±0.150	0.862±0.122

表5 DGA不同处理时间对TGF-β1诱导的NRK-49F及NRK-52E细胞活力的影响

Tab.5 Effect of different treatment durations with DGA on viability of TGF-β1-induced NRK-49F and NRK-52E (Mean±SD, n=3)

Group	Induction time NRK-49F		Induction time NRK-52E
Blank	1.711±0.053		1.819±0.031
Control	2.093±0.063		2.219±0.041
DGA	0 h	2.101±0.032	0 h	2.108±0.027*
	24 h	2.082±0.113	12 h	2.052±0.084*
	48 h	1.974±0.049*	24 h	1.943±0.039^#

图10 参芪泄浊饮含药血清及DGA对TGF-β1诱导的NRK-49F及NRK-52E细胞α-SMA、Col-I、E-Cadherin表达的影响

Fig.10 Effects of SQXZD-medicated serum and DGA on expression of α-SMA, Col-I, and E-cadherin in TGF-β1-induced NRK-49F (A) and NRK-52E cells (B). **P<0.01 vs Control group; *P<0.05, #P<0.01 vs Model group; ▲P<0.05 vs DGA group. n=3.

图11 参芪泄浊饮含药血清及DGA对TGF-β1诱导的NRK-49F及NRK-52E细胞自噬荧光的影响

Fig.11 Effects of SQXZD-medicated serum and DGA on autophagic fluorescence in TGF-β1-induced NRK-49F cells (A, C) and NRK-52E cells (B, D) (×400). **P<0.01 vs Control group; *P<0.05, #P<0.01 vs Model group; ▲P<0.05 vs DGA group. n=3.

图12 参芪泄浊饮含药血清及DGA对TGF-β1诱导的NRK-49F及NRK-52E细胞LC3B-II、p62/SQSTM1表达的影响

Fig.12 Effects of SQXZD-medicated serum and DGA on the expression of LC3B-II and p62/SQSTM1 in TGF-β1-induced NRK-49F (A) and NRK-52E cells (B). **P<0.01 vs Control group; *P<0.05, #P<0.01 vs Model group; ▲P<0.05 vs DGA group. n=3.

图13 参芪泄浊饮含药血清及DGA对TGF-β1诱导的NRK-49F及NRK-52E细胞超微结构的影响

Fig.13 Effects of SQXZD-medicated serum and DGA on ultrastructure of TGF-β1-induced NRK-49F (A) and NRK-52E cells (B) (Transmission electron microscopy, ×12 000), red arrows indicate autophagosomes.

图14 参芪泄浊饮含药血清及DGA对TGF-β1诱导的NRK-49F及NRK-52E细胞p-ERK1/2/ERK1/2、p-mTOR/mTOR表达的影响

Fig.14 Effects of SQXZD-medicated serum and DGA on expression of p-ERK1/2/ERK1/2 and p-mTOR/mTOR in TGF-β1-induced NRK-49F (A) and NRK-52E cells (B). **P<0.01 vs Control group; *P<0.05, #P<0.01 vs Model group; ▲P<0.05 vs DGA group. n=3.

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