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Nrf2 knockout attenuates the astragaloside IV therapeutic effect on kidney fibrosis from liver cancer by regulating pSmad3C/3L pathways

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Abstract

Fibrotic kidney injury from hepatocarcinogenesis seriously impacts treatment effect. Astragaloside IV (AS-IV), an extract of Astragalus membranaceus, has several pharmacological activities, which are useful in the treatment of edema and fibrosis. Nrf2/HO-1 is a key antioxidant stress pathway and help treatment of kidney injury. Smad3 phosphorylation is implicated in hepatocarcinogenesis. Our previous study clarified that Smad3 is differentially regulated by different phosphorylated forms of Smad3 on hepatocarcinogenesis. Therefore, we investigated the contribution of AS-IV on the therapy of kidney fibrosis from hepatocarcinogenesis. And the focus was on whether the phosphorylation of Smad3 and the regulation of Nrf2/HO-1 pathway were involved during AS-IV therapy and whether there is an effect of Nrf2 knockout on the phosphorylation of Smad3. We performed TGF-β1 stimulation on HK-2 cells and intervened with AS-IV. Furtherly, we investigated renal injury of AS-IV on Nrf2 knockout mice during hepatocarcinogenesis and its mechanism of action. On the one hand, in vitro results showed that AS-IV reduced the ROS and α-SMA expression of HK-2 by promoting the expression pSmad3C/p21 of and Nrf2/HO-1 and suppressed the expression of pSmad3L/PAI-1. On the other hand, the in vivo results of histopathological features, serological biomarkers, and oxidative damage indicators showed that Nrf2 knockout aggravated renal injury. Besides, Nrf2 deletion decreased the nephroprotective effect of AS-IV by suppressing the pSmad3C/p21 pathway and promoting the pSmad3L/PAI-1 pathway. The experimental results were as we suspected. And we identify for the first time that Nrf2 deficiency increases renal fibrosis from hepatocarcinogenesis and attenuates the therapeutic effects of AS-IV via regulating pSmad3C/3L signal pathway.

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Data availability

The data and materials that support the findings of this study are available from the corresponding author upon reason able request.

Abbreviations

AS-IV:

Astragaloside IV

BUN:

Blood urea nitrogen

CCl4 :

Carbon tetrachloride

DEN:

Diethylinitrosamine

DMSO:

Dimethyl sulfoxide

HCC:

Hepatocellular carcinoma

HE:

Hematoxylin and eosin

HRS:

Hepatorenal syndrome

HO-1:

Heme oxygenase-1

Masson:

Masson-trichrome staining

MDA:

Malondialdehyde

Nrf2:

Nuclear factor erythroid 2-related factor 2

NS:

Normal saline

PAI-1:

Plasminogen activator inhibitor-1

pSmad3C:

C-terminally phosphorylated Smad3

pSmad3L:

Linker-phosphorylated Smad3

Scr:

Serum creatinine

SOD:

Superoxide dismutase

TGF-β1:

Transforming growth factor-beta 1

TβRI:

TGF-β type I receptor

α-SMA:

Alpha smooth muscle actin

β-actin:

Beta-actin (β-non-muscle)

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Acknowledgements

We would like to thank all members of the Research Center of Anhui Medical University for their effective techniques and valuable help during the experiments. We thank Dr. K. Matsuzaki for presenting us with rabbit polyclonal anti-pSmad3L (Ser208/213) antibody. We thank Professor Xiaoming Meng for the gift of HK-2 cells.

Funding

This study was supported by the National Natural Science Foundation of China (81874354, 82074073).

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Author notes

  1. Qin Wang and Jiacheng Xu contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, School of Basic Medical Sciences, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China

    Qin Wang, Jiacheng Xu, Miaomiao Li, Yuqing Chen, Yingying Xu, Lili Li, Yongfang Gong & Yan Yang

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  1. Qin Wang
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Contributions

QW and XJC conceived and designed the research. QW conducted experiments and wrote the manuscript. MM L and YQ C contributed to new reagents and analytical tools. YY X, LL L and YF G provided the methodology and analyzed data. YY were responsible for the supervision and funding acquisition. The authors declare that all data were generated in-house and that no paper mill was used.

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Correspondence to Yan Yang.

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The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Animal Ethics Committee of Anhui Medical University (Ethics approval code: LLSC, 20221064, Approval Date: 2 June 2022).

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Wang, Q., Xu, J., Li, M. et al. Nrf2 knockout attenuates the astragaloside IV therapeutic effect on kidney fibrosis from liver cancer by regulating pSmad3C/3L pathways. Naunyn-Schmiedeberg's Arch Pharmacol 397, 1687–1700 (2024). https://doi.org/10.1007/s00210-023-02711-2

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