Curcumol alleviates liver fibrosis by inducing endoplasmic reticulum stress-mediated necroptosis of hepatic stellate cells through Sirt1/NICD pathway

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Biochemistry, Biophysics and Molecular Biology

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Introduction

Materials and Methods

Reagents and antibodies

Animal procedures and treatments

Histological analysis

Cell culture

Trypan blue staining

Cell viability assay

SiRNA transfection

Real-time PCR

Western blot analysis

Immunofluorescence analysis

Transmission electron microscopy (TEM)

Co-Immunoprecipitation (Co-IP) experiment

Determination of acetylation levels

Statistical analysis

Results

Curcumol attenuates liver fibrosis by inducing necroptosis of HSCs

Curcumol induces ER stress in HSCs

ER stress inhibitor impairs the induction effect of curcumol on HSCs necroptosis

ER stress regulates curcumol-induced necroptosis through Sirt1 in HSCs

Sirt1-mediated deacetylation of NICD is required for curcumol-induced necroptosis in HSCs

Curcumol alleviates liver fibrosis through Sirt1 in vivo

Discussion

Conclusions

Supplemental Information

Curcumol reduces liver fibrosis through Sirt1-mediated necroptosis in vivo.

(A) The liver index (liver weight/body weight) of mice. (B) Immunohistochemical staining of α-SMA.

DOI: 10.7717/peerj.13376/supp-1

Curcumol alleviates liver fibrosis by inducing endoplasmic reticulum stress-mediated necroptosis of hepatic stellate cells through Sirt1/NICD pathway.

Curcumol induces ER stress in HSCs, and ER stress-induced cellular dysfunction causes necroptosis in HSCs. The specific mechanism is through activation of the downstream Sirt1/Notch signaling pathway. Sirt1-mediated deacetylation of the intracellular domain of Notch (NICD) and promotes NICD degradation. The inhibition of Notch signaling pathway contributes to hepatic stellate cell necroptosis and alleviates liver fibrosis.

DOI: 10.7717/peerj.13376/supp-2

Repeated results of all corresponding Western Blot results.

DOI: 10.7717/peerj.13376/supp-3

The Corresponding qRT-PCR and MTT results.

DOI: 10.7717/peerj.13376/supp-4

Additional Information and Declarations

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Sumin Sun conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Sheng Huan performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Zhanghao Li performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Yue Yao performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Ying Su performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Siwei Xia performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Shijun Wang conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Xuefen Xu conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Jiangjuan Shao conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Zili Zhang conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Feng Zhang conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Jinbo Fu conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Shizhong Zheng conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

All experimental procedures were approved by the Nanjing University of Chinese Medicine (Nanjing, China) institutional and local animal care and use committees. All of the animals were cared for humanely, according to National Institutes of Health guidelines.

Data Availability

The following information was supplied regarding data availability:

The raw measurements are available in the Supplemental Files.

Funding

The work was supported by the National Natural Science Foundation of China (82073914, 82000572, 81870423), the Natural Science Foundation of Jiangsu Province (BK20200840), the Major Project of the Natural Science Research of Jiangsu Higher Education Institutions (19KJA310005), the General Projects of the Natural Science Research of Jiangsu Higher Education Institutions (20KJB310003), the Joint Project of Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica and Yangtze River Pharmaceutical (JKLPSE202005), the Natural Science Foundation of Nanjing University of Chinese Medicine (NZY82000572), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX21_1732, KYCX21_1637 and SJCX20_0569), and the Open Project of Chinese Materia Medica First-Class Discipline of Nanjing University of Chinese Medicine (2020YLXK022 and 2020YLXK023). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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