Available online 29 February 2024

Author links open overlay panel, , , , , , AbstractBackground

Calcitriol has the potential to counteract fibrotic diseases beyond its classical action of maintaining calcium and bone metabolism; however, its functional mechanism remains unknown. Autophagy-related gene 16-like 1 (Atg16l1) is one of the genes related to autophagy and is involved in protecting against fibrotic diseases. The present study aimed to explore the contribution of autophagy to the inhibition of calcitriol-induced hepatic fibrosis, as well as its potential molecular mechanism.

Methods

Carbon tetrachloride (Ccl4)-treated mice were established as hepatic fibrosis models and received calcitriol treatment for 6 weeks. Quantification of Sirius red staining and measurement of key fibrotic markers (collagen-1 and α-SMA) was performed to detect hepatic fibrosis. Chloroquine (CQ) treatment was used to observe autophagic flux, and 3-methyladenine (3-MA) was used to inhibit autophagy. Furthermore, the effects of calcitriol ontransforming growth factor β1 (TGFβ1)-stimulated primary hepatic stellate cells (HSCs) were detected. Downregulation of Atg16l1 or vitamin D receptor (VDR) in LX-2 cells was used to explore the mechanism of action of calcitriol in fibrosis and autophagy. Additionally, the electrophoretic mobility shift assay (EMSA) was used to investigate the interactions between VDR and ATG16L1.

Results

Calcitriol increased the expression of VDR and ATG16L1, enhanced autophagy and attenuated hepatic fibrosis. 3-MA treatment and VDR silencing abolished the protective effects of calcitriol against fibrosis. Calcitriol-induced anti-fibrosis effects were blocked by ATG16L1 suppression. Furthermore, VDR bound to the ATG16L1 promoter and downregulation of VDR decreased the expression of ATG16L1 in LX-2 cells.

Conclusion

Calcitriol mitigates hepatic fibrosis partly through ATG16L1-mediated autophagy.

Section snippetsINTRODUCTION

Progressive hepatic fibrosis is one of the important public health problems worldwide, causing serious clinical consequences such as portal hypertension, hepatic dysfunction, and hepatocellular carcinoma.1,2 Liver fibrosis is a type of irregular tissue-repairing response to chronic hepatic injuries induced mainly by a virus infection, autoimmune disorders, and exposure to drugs or toxic compounds.3,4 Liver fibrosis is characterized by excessive accumulation of extracellular matrix (ECM)

Animals and treatment

All animal experiments in this study were in accordance with the National Institutes of Health guidelines and were approved by the ethical criteria of the Animal Care and Use Committee of Huazhong University of Science and Technology. Eight-week-old male C57BL/6 mice were obtained from the Wuhan University Center for Animal Experiments (Wuhan, China) and bred under standard conditions. To establish a liver fibrosis model, mice were intraperitoneally (IP) injected with carbon tetrachloride (Ccl4

Calcitriol attenuated hepatic fibrosis in vivo andin vitro

To assess whether calcitriol attenuates hepatic fibrogenesis in vivo, a mouse model of liver fibrosis was established by treatment with Ccl4, a widely used hepatotoxic agent. Liver fibrosis manifested as substantial collagen accumulation, and Ccl4/calcitriol-co-treated mice showed a significant reduction in liver fibrosis, as evidenced by a reduction in the Sirius red-positive area in the liver (Fig. 1A and B). Measurement of key fibrotic marker genes such as collagen-1, TGFβ1, and Timp1 were

DISCUSSION

Calcitriol modulates hundreds of different genes and has the potential to counteract fibrotic diseases in addition to its classical action of maintaining calcium and bone metabolism. Autophagy is critically implicated in the progression of hepatology and is expected to be an attractive target for antifibrotic therapies. Therefore, the identification of explicit antifibrotic mechanisms may provide a rationale for controlling hepatic fibrosis. In the present study, we demonstrated that calcitriol

CONCLUSION

In summary, we demonstrated that calcitriol exhibits an anti-fibrotic effect on hepatic fibrosis byregulating VDR expression in Ccl4-induced mice and in TGFβ1-stimulated LX-2 cells and HSCs. This protective effect is dependent on the induction of autophagy by ATG16L1. These insights reveal a central role for calcitriol in liver fibrosis and may provide a rational approach for liver fibrosis treatment.

AUTHOR CONTRIBUTIONS

ESG, HXY, RLL, JKY performed research, collected and analyzed data; HXYand SPL prepared figures, analyzed and interpreted the data; ESG, ADL and XJJ wrote paper; ADL and XJJ conceived the study, designed research, revised the paper and discussed analyses; All authors have read and approved the final manuscript.

SOURCE OF FUNDING

This project was by grants from the National Natural Science Fund of China (NSFC) (81171586, 81300343), Natural Science Fund of Henan Province (212300410266) and Medical Science and Technology Research Project of Henan Province (2018020101).

DATA AVAILABILITY

All data generated or analyzed during this study are included in this article.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Animal experiments were conducted under the approval of the ethical criteria of the Animal Care and Use Committee of Huazhong University of Science and Technology, and in strict accordance with the National Institutes of Health guidelines. All efforts were made to minimize the number and suffering of the included animals.

ABBREVIATIONS

ATG16L1: autophagy-related gene 16-like 1; Ccl4: carbon tetrachloride; ECM: extracellular matrix; HSCs: hepatic stellate cells; TGFβ1: transforming growth factor β1; VDR: vitamin D receptor; VDRE: vitamin D response element; IP: intraperitoneally; 3-MA: 3-methyladenine; CQ: chloroquine; H&E: hematoxylin-eosin; DMEM: Dulbecco's modified Eagle's medium; MOIs: multiplicities of infection; PBS: phosphate-buffered saline; DAPI: 4′,6-diamidino-2-phenylindole; α-SMA: smooth muscle actin; ATG7:

Declaration of competing interest

The authors declare no conflict of interests.

ACKNOWLEDGMENT

Funding from Sysmex Corp. is gratefully acknowledged.

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© 2024 Published by Elsevier Inc. on behalf of Southern Society for Clinical Investigation.