Bushen Tongluo recipe (补肾通络方) improves oxidative stress homeostasis, inhibits transforming growth factor/Notch signaling pathway, and regulates the lncRNA maternally expressed gene 3/miR-145 axis to delay diabetic kidney disease

doi:10.19852/j.cnki.jtcm.2025.03.011

Abstract

Abstract:

OBJECTIVES: To investigate the effect of Bushen Tongluo recipe (BSTLR, 补肾通络方) on rats with diabetic kidney disease (DKD) and to explore the underlying mechanism of action.
METHODS: The rat model of DKD was established, and rats were treated with different doses of BSTLR. Body weight and the levels of urinary protein, α1-microglobulin, glucose, blood urea nitrogen, creatinine, Cystatin C, superoxide dismutase, malondialdehyde, and catalase were analyzed biochemically or by enzyme-linked immunosorbent assay. The pathological damage to renal tissues was assessed by hematoxylin-eosin staining. Immunohistochemical staining was carried out to detect the expression levels of fibronectin, E-cadherin, α-smooth muscle actin, laminin, vimentin, collagen type Ⅳ in kidney tissues. Western blot analysis was conducted to analyze the expression levels of Nephrin, Desmin, Podocin, transforming growth factor-β1, mothers against decapentaplegic homolog 3 (Smad3), Notch1, jagged, hairy and enhancer of split 1 (Hes1) in kidney tissues, and the expression levels of maternally expressed gene 3 (MEG3) and miR-145 were measured by quantitative reverse transcription-polymerase chain reaction. Moreover, dual-luciferase reporter assay was employed to verify the binding of miR-145 to MEG3.
RESULTS: BSTLR increased the body weight of DKD rats, effectively ameliorated the renal function and pathological injury in DKD, regulated the balance of renal oxidative stress, inhibited the TGF/Notch signaling pathway, and affected the variations in the lncRNA MEG3/miR-145 axis.
CONCLUSION: BSTLR improved oxidative stress homeostasis, inhibited the TGF/Notch signaling pathway, and regulated the lncRNA MEG3/miR-145 axis, effectively delaying the progression of DKD.

Key words: diabetic nephropathies, oxidative stress, transforming growth factors, receptors, Notch, signal transduction, RNA, long noncoding, maternally expressed gene 3, miR-145, Bushen Tongluo recipe

XU Bojun, TAO Tian, ZHAO Liangbin, ZHENG Hui, ZHAN huakui, GUO Julan. Bushen Tongluo recipe (补肾通络方) improves oxidative stress homeostasis, inhibits transforming growth factor/Notch signaling pathway, and regulates the lncRNA maternally expressed gene 3/miR-145 axis to delay diabetic kidney disease[J]. Journal of Traditional Chinese Medicine, 2025, 45(3): 561-570.

Figures/Tables 5

Table 1 Body weight and kidney coefficient ($\bar{x}±s$)

Group	n	IBW (g)	STZ 3 d body weight (g)	High lipid/treatment body weight (g)			KC (%)
Group	n	IBW (g)	STZ 3 d body weight (g)	1W	5W	10W	KC (%)
Control	5	279.924±1.708	349.502±11.552	379.500±9.649	499.500±11.032	569.500±4.155	0.568±0.013
Model	5	279.588±2.859	272.386±5.211^a	279.166±4.383^a	288.212±4.359^a	317.516±4.161^a	1.432±0.058^a
Losartan	5	279.733±3.515	324.003±3.574	324.201±3.749^b	379.613±4.409^b	430.276±4.948^b	0.868±0.024^b
BSTLR-L	5	280.300±3.827	279.638±4.604	299.086±4.416^b	356.498±3.511^b	392.542±3.750^b	1.025±0.013^b
BSTLR-M	5	279.690±1.801	290.316±5.448^b	309.722±5.132^b	375.180±4.152^b	425.740±5.422^b	0.885±0.015^b
BSTLR-H	5	280.020±2.505	308.908±4.851^b	329.892±4.663^b	394.546±4.077^b	444.170±5.454^b	0.761±0.025^b

Figure 1 Effect of BSTLR on renal function in DKD rats A: concentrations of UP, α1-MG, GLU, BUN, CREA, and CysC in rats of 1 week; B: concentrations of UP, α1-MG, GLU, BUN, CREA, and CysC in rats of 5 week; C: concentrations of UP, α1-MG, GLU, BUN, CREA, and CysC in rats of 10 week; A1, B1, C1: UP; A2, B2, C2: α1-MG; A3, B3, C3: GLU; A4, B4, C4: BUN; A5, B5, C5: CREA; A6, B6, C6: CysC. Control and Model groups were gavaged with equal amounts of distilled water, the Losartan group was gavaged with Losartan (50 mg/kg), and the BSTLR-L, BSTLR-M, and BSTLR-H groups were gavaged with BSTLR at concentrations of 1.2, 2.4, and 4.8 g/mL, respectively, once a day. BSTLR: Bushen Tongluo recipe; DKD: diabetic kidney disease; UP: urinary protein; α1-MG: α1-microglobulin; GLU: glucose; BUN: blood urea nitrogen; CREA: creatinine; CysC: cystatin C. Data were represented as mean ± standard deviation (n = 5). Statistical differences between groups were determined by one-way analysis of variance. Compared with control group, aP < 0.05; compared with model group, bP < 0.05.

Figure 2 Effect of BSTLR on renal injury in DKD rats A: HE staining of kidney tissues; A1: Control (× 100, scale bar, 200 μm) ; A2: Model (× 100, scale bar, 200 μm); A3: Losartan (× 100, scale bar, 200 μm); A4: BSTLR-L (× 100, scale bar, 200 μm); A5: BSTLR-M (× 100, scale bar, 200 μm); A6: BSTLR-H (× 100, scale bar, 200 μm); A7: Control (× 400, scale bar, 50 μm); A8: Model (× 400, scale bar, 50 μm); A9: Losartan (× 400, scale bar, 50 μm); A10: BSTLR-L (× 400, scale bar, 50 μm); A11: BSTLR-M (× 400, scale bar, 50 μm); A12: BSTLR-H (× 400, scale bar, 50 μm). B: activities of MDA, SOD, and CAT in kidney tissues; B1: SOD; B2: MDA; B3: CAT; C: expression levels of Nephrin, Desmin, and Podocin in kidney tissues; C1: protein band; C2: Nephrin; C3: Desmin; C4: Podocin. Control and Model groups were gavaged with equal amounts of distilled water, the Losartan group was gavaged with Losartan (50 mg/kg), and the BSTLR-L, BSTLR-M, and BSTLR-H groups were gavaged with BSTLR at concentrations of 1.2, 2.4, and 4.8 g/mL, respectively, once a day. BSTLR: Bushen Tongluo recipe; DKD: diabetic kidney disease; SOD: superoxide dismutase; MDA: malondialdehyde; CAT: catalase. Data were represented as mean ± standard deviation (n = 5). Statistical differences between groups were determined by one-way analysis of variance. Compared with control group, aP < 0.05; compared with model group, bP < 0.05.

Figure 3 Effect of BSTLR on renal injury in DKD rats A: immunohistochemical staining of FN (× 400, scale bar, 50 μm) in kidney tissues. B: immunohistochemical staining of E-cad (× 400, scale bar, 50 μm) in kidney tissues. C: immunohistochemical staining of α-SMA (×400, scale bar, 50 μm) in kidney tissues. D: immunohistochemical staining of LN (× 400, scale bar, 50 μm) in kidney tissues. E: immunohistochemical staining of Vim (× 400, scale bar, 50 μm) in kidney tissues. F: immunohistochemical staining of Col Ⅳ (× 400, scale bar, 50 μm) in kidney tissues. A1, B1, C1, D1, E1, F1: Control; A2, B2, C2, D2, E2, F2: Model; A3, B3, C3, D3, E3, F3: Losartan; A4, B4, C4, D4, E4, F4: BSTLR-L; A5, B5, C5, D5, E5, F5: BSTLR-M; A6, B6, C6, D6, E6, F6: BSTLR-H; G: positive expression levels of FN; H: positive expression levels of E-cad; I: positive expression levels of α-SMA; J: positive expression levels of LN; K: positive expression levels of Vim; L: positive expression levels of Col IV. Control and Model groups were gavaged with equal amounts of distilled water, the Losartan group was gavaged with Losartan (50 mg/kg), and the BSTLR-L, BSTLR-M, and BSTLR-H groups were gavaged with BSTLR at concentrations of 1.2, 2.4, and 4.8 g/mL, respectively, once a day. BSTLR: Bushen Tongluo recipe; DKD: diabetic kidney disease; FN: fibronectin; E-cad: E-cadherin; α-SMA: α-smooth muscle actin; LN: laminin; Vim: vimentin; Col IV: collagen type Ⅳ. Data were represented as mean ± standard deviation (n = 5). Statistical differences between groups were determined by one-way analysis of variance. Compared with the control group, aP < 0.05; compared with the model group, bP < 0.05.

Figure 4 Effect of BSTLR on renal TGF/Notch pathway in DKD rats A: expression levels of TGF-β1 in kidney tissues; B: expression levels of Smad3 in kidney tissues; C: expression levels of Notch1 in kidney tissues; D: expression levels of Jagged1 in kidney tissues; E: expression levels of Hes1 in kidney tissues; F: protein band. Control and Model groups were gavaged with equal amounts of distilled water, the Losartan group was gavaged with Losartan (50 mg/kg), and the BSTLR-L, BSTLR-M, and BSTLR-H groups were gavaged with BSTLR at concentrations of 1.2, 2.4, and 4.8 g/mL, respectively, once a day. BSTLR: Bushen Tongluo recipe; DKD: diabetic kidney disease; TGF-β1: transforming growth factor-β1; Smad3: mothers against decapentaplegic homolog 3; Hes1: hairy and enhancer of split 1. Data were represented as mean ± standard deviation (n = 5). Statistical differences between groups were determined by one-way analysis of variance. Compared with the control group, aP < 0.05; compared with the model group, bP < 0.05.

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