Sanqi Oral Solution Ameliorates Renal Ischemia/Reperfusion Injury via Reducing Apoptosis and Enhancing Autophagy: Involvement of ERK/mTOR Pathways

Abstract

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI) is a significant health problem with high morbidity and mortality, yet prophylaxis strategies and effective drugs are limited. Sanqi oral solution (SQ) is a formulated medicine widely used in clinical settings to treat various renal diseases via enriching qi and activating blood circulation while its role on I/R-AKI remains unclear. Herein, by establishing rat I/R-AKI models, we intended to investigate the effect of SQ on the prevention of I/R-AKI and explore its underlying mechanisms. We demonstrated that SQ treatment significantly attenuated renal dysfunction of I/R-AKI, alleviated histological damages, inhibited renal apoptosis, and enhanced autophagy. Further investigation proved that SQ could significantly inhibit the activation of ERK and mTOR signaling pathways. Moreover, its renoprotective effect can be abolished by autophagy inhibitor 3-methyladenine (3-MA). Collectively, our results suggest that SQ exerts renoprotective effects on renal I/R injury via reducing apoptosis and enhancing autophagy, which are associated with regulating ERK/mTOR pathways.

Keywords: Radix Astragali; Radix Notoginseng; acute kidney injury (AKI); apoptosis; autophagy; extracellular signal-regulated kinase (ERK); mammalian target of rapamycin (mTOR); renal ischemia-reperfusion (I/R).

Figure 1

SQ attenuates I/R-induced renal dysfunction and histopathological changes. (A) Representative HE staining of renal tissues. (B) Jablonski grade to reflect tubular injury calculated from HE staining. (C) BUN and SCR levels which indicated the function of kidney filtration. Data were presented as means ± SD, **p < 0.01 and ***p < 0.001 versus Sham group, ^###p < 0.001 versus I/R group. n = 6 in each group.

Figure 2

SQ reduces apoptosis in I/R-AKI. (A) Representative western blot of apoptosis-related proteins: Bcl-2, Bax, and cleaved Caspase 3. (B) Quantitative analysis of western blot results. β-actin was used as loading control. (C) Representative images of TUNEL assay in each group. (D) Representative images of immunohistochemical assay of cleaved Caspase 3 expression in each group. (E) Quantitative analysis of TUNEL and immunohistochemical stainings. Data were presented as means ± SD, *p < 0.05 and ***p < 0.001, versus Sham group, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus I/R group. n = 3 in each group.

Figure 3

SQ enhances autophagy in IR-AKI. Representative western blot of autophagy markers: Beclin1, SQSTM1/p62, and LC3, and the corresponding quantitative analysis. β-actin was used as loading control. Data were presented as means ± standard deviation (SD), *p < 0.05 and ***p < 0.001 versus Sham group, ^#p < 0.05 and ^###p < 0.001 versus I/R group. n = 3 in each group.

Figure 4

Effects of SQ on ERK and mTOR signaling pathways in I/R-AKI. Representative western blot of p-ERK, ERK, p-mTOR, and mTOR, and the corresponding quantitative analysis. Data were presented as means ± SD, *p < 0.05 and ***p < 0.001 versus Sham group, ^#p < 0.05 and ^##p < 0.01 versus I/R group. n = 3 in each group.

Figure 5

3-MA suppresses autophagy in I/R-AKI rats with SQ treatment. Representative western blot of PI3K III related autophagy protein SQSTM1/p62 and LC3, and the corresponding quantitative analyses. β-actin was used as loading control. Data were presented as means ± SD, *p < 0.05 and ***p < 0.001 versus Sham group, ^##p < 0.01 versus I/R group, ^p < 0.05, ^^p < 0.01 versus I/R+SQ-H group. n = 3 in each group.

Figure 6

3-MA aggravates renal damages in I/R-AKI rats with SQ treatment. (A) Representative HE staining of kidney tissues. (B) Jablonski grade to reflect tubular injury calculated from HE staining. (C) BUN and SCR to indicate the function of kidney filtration. Data were presented as means ± SD, **p < 0.01, and ***p < 0.001 versus Sham group, ^###p < 0.001 versus I/R group, ^p < 0.05 versus I/R+SQ-H group. n = 6 in each group.

Figure 7

3-MA enhances apoptosis in I/R-AKI rats with SQ treatment. (A) Representative western blot of apoptosis-related protein Bcl-2, Bax, Caspase 3 (B) Quantitative analysis of western blot results. β-actin was used as loading control. (C) Representative images of TUNEL assay in each group. (D) Representative images of immunohistochemical assay of cleaved Caspase 3 expression in each group. (E) Quantitative analysis of TUNEL and immunohistochemical stainings. Data were presented as means ± SD, *p < 0.05, **p < 0.01, and ***p < 0.001 versus Sham group, ^##p < 0.01, and ^###p < 0.001 versus I/R group. ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 versus I/R+SQ-H group. n = 3 in each group.