Shenqi Xiezhuo Decoction ameliorates renal fibrosis in rats by inhibiting excessive autophagy via modulating the ERK1/2-mTOR signaling pathway

doi:10.12122/j.issn.1673-4254.2026.10.03

Abstract

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

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.

Figures/Tables 17

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

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

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

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.

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

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.

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.

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.

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

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*

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

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^#

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.

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.

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.

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.

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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