Dihydroartemisinin inhibits the activation and proliferation of hepatic stellate cells by regulating miR‑29b‑3p

Huan,Sheng; Huan,Sheng; Sun,Sumin; Sun,Sumin; Song,Shilian; Song,Shilian; Dai,Jin; Dai,Jin; Zhu,Guining; Zhu,Guining; Zhong,Yanling; Zhong,Yanling; Ji,Yihao; Ji,Yihao; Zheng,Shizhong; Zheng,Shizhong; Yin,Guoping; Yin,Guoping

doi:10.3892/ijmm.2023.5243

Dihydroartemisinin inhibits the activation and proliferation of hepatic stellate cells by regulating miR‑29b‑3p

Authors:
- Sheng Huan
- Sumin Sun
- Shilian Song
- Jin Dai
- Guining Zhu
- Yanling Zhong
- Yihao Ji
- Shizhong Zheng
- Guoping Yin
View Affiliations

Affiliations: Department of Anesthesiology, The Second Hospital of Nanjing, Nanjing, Jiangsu 210037, P.R. China, Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210046, P.R. China, Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210046, P.R. China
Published online on: April 6, 2023 https://doi.org/10.3892/ijmm.2023.5243
Article Number: 40
Copyright: © Huan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Liver fibrosis is an early pathological feature of hepatic diseases. Hepatic stellate cell (HSC) activation and disordered proliferation are associated with liver fibrosis. The present study identified significant differences in the expression levels of microRNA (miRNA/miR)‑29b‑3p in clinical samples and multiple miRNA databases. Subsequently, the specific antifibrotic mechanism of miR‑29b‑3p was further elucidated. Reverse transcription‑quantitative PCR, western blot, ELISA and immunofluorescence were used to detect the expression levels of target genes and proteins. Oil red O, Nile red and trypan blue staining were used to evaluate HSC activation and cell viability. A luciferase assay was used to detect the relationship between miR‑29b‑3p and VEGFA. Adhesion, wound healing, apoptosis double staining and JC‑1 assays were used to detect the effects of VEGFR1 and VEGFR2 knockdown on HSCs. Immunoprecipitation and fluorescence colocalization were used to identify interactions between the proteins. Furthermore, a rat fibrosis model was constructed to investigate the effects of dihydroartemisinin (DHA) and miR‑29b‑3p in vivo and in vitro. The results indicated that miR‑29b‑3p both inhibited the activation of HSCs and limited the proliferation of activated HSCs via lipid droplet recovery and VEGF pathway regulation. VEGFA was identified as a direct target of miR‑29b‑3p, and knockdown of VEGFA induced cell apoptosis and autophagy. Notably, VEGFR1 and VEGFR2 knockdown both promoted apoptosis; however, VEGFR1 knockdown inhibited autophagy, whereas VEGFR2 knockdown induced autophagy. Furthermore, it was revealed that VEGFR2 regulated autophagy by mediating the PI3K/AKT/mTOR/ULK1 pathway. VEGFR2 knockdown also led to ubiquitination of heat shock protein 60, ultimately inducing mitochondrial apoptosis. Finally, DHA was identified as a natural agonist of miR‑29‑3p that effectively prevented liver fibrosis in vivo and in vitro. Overall, the present study determined the molecular mechanism by which DHA inhibited HSC activation and prevented liver fibrosis.

View Figures

View References

1	Kisseleva T and Brenner D: Molecular and cellular mechanisms of liver fibrosis and its regression. Nat Rev Gastroenterol Hepatol. 18:151–166. 2021. View Article : Google Scholar
2	Hernandez-Gea V and Friedman SL: Pathogenesis of liver fibrosis. Annu Rev Pathol. 6:425–456. 2011. View Article : Google Scholar
3	Parola M and Pinzani M: Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues. Mol Aspects Med. 65:37–55. 2019. View Article : Google Scholar
4	Kang N, Gores GJ and Shah VH: Hepatic stellate cells: Partners in crime for liver metastases? Hepatology. 54:707–713. 2011. View Article : Google Scholar : PubMed/NCBI
5	Trivedi P, Wang S and Friedman SL: The power of plasticitymetabolic regulation of hepatic stellate cells. Cell Metab. 33:242–257. 2021. View Article : Google Scholar
6	Rupaimoole R and Slack FJ: MicroRNA therapeutics: Towards a new era for the management of cancer and other diseases. Nat Rev Drug Discov. 16:203–222. 2017. View Article : Google Scholar : PubMed/NCBI
7	Krol J, Loedige I and Filipowicz W: The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet. 11:597–610. 2010. View Article : Google Scholar : PubMed/NCBI
8	Dong H, Lei J, Ding L, Wen Y, Ju H and Zhang X: MicroRNA: Function, detection, and bioanalysis. Chem Rev. 113:6207–6233. 2013. View Article : Google Scholar : PubMed/NCBI
9	Kim VN: MicroRNA biogenesis: Coordinated cropping and dicing. Nat Rev Mol Cell Biol. 6:376–385. 2005. View Article : Google Scholar : PubMed/NCBI
10	Su T, Xiao Y, Xiao Y, Guo Q, Li C, Huang Y, Deng Q, Wen J, Zhou F and Luo XH: Bone marrow mesenchymal stem cells-derived exosomal MiR-29b-3p regulates aging-associated insulin resistance. ACS Nano. 13:2450–2462. 2019.PubMed/NCBI
11	Xue Y, Fan X, Yang R, Jiao Y and Li Y: miR-29b-3p inhibits post-infarct cardiac fibrosis by targeting FOS. Biosci Rep. 40:BSR202012272020. View Article : Google Scholar : PubMed/NCBI
12	Novo E, Cannito S, Zamara E, Valfrè di Bonzo L, Caligiuri A, Cravanzola C, Compagnone A, Colombatto S, Marra F, Pinzani M and Parola M: Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells. Am J Pathol. 170:1942–1953. 2007. View Article : Google Scholar : PubMed/NCBI
13	Sun J, Shi L, Xiao T, Xue J, Li J, Wang P, Wu L, Dai X, Ni X and Liu Q: microRNA-21, via the HIF-1α/VEGF signaling pathway, is involved in arsenite-induced hepatic fibrosis through aberrant cross-talk of hepatocytes and hepatic stellate cells. Chemosphere. 266:1291772021. View Article : Google Scholar
14	Huang YH and Yeh CT: Functional Compartmentalization of HSP60-Survivin interaction between mitochondria and cytosol in cancer cells. Cells. 9:232019. View Article : Google Scholar : PubMed/NCBI
15	Zhang Z, Yao Z, Zhao S, Shao J, Chen A, Zhang F and Zheng S: Interaction between autophagy and senescence is required for dihydroartemisinin to alleviate liver fibrosis. Cell Death Dis. 8:e28862017. View Article : Google Scholar : PubMed/NCBI
16	Chen Q, Chen L, Kong D, Shao J, Wu L and Zheng S: Dihydroartemisinin alleviates bile duct ligation-induced liver fibrosis and hepatic stellate cell activation by interfering with the PDGF-βR/ERK signaling pathway. Int Immunopharmacol. 34:250–258. 2016. View Article : Google Scholar : PubMed/NCBI
17	Chen Q, Chen L, Wu X, Zhang F, Jin H, Lu C, Shao J, Kong D, Wu L and Zheng S: Dihydroartemisinin prevents liver fibrosis in bile duct ligated rats by inducing hepatic stellate cell apoptosis through modulating the PI3K/Akt pathway. IUBMB Life. 68:220–231. 2016. View Article : Google Scholar : PubMed/NCBI
18	Seeliger D and de Groot BL: Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des. 24:417–422. 2010. View Article : Google Scholar : PubMed/NCBI
19	Murakami Y, Toyoda H, Tanahashi T, Tanaka J, Kumada T, Yoshioka Y, Kosaka N, Ochiya T and Taguchi YH: Comprehensive miRNA expression analysis in peripheral blood can diagnose liver disease. PLoS One. 7:e483662012. View Article : Google Scholar : PubMed/NCBI
20	Vuppalanchi R, Liang T, Goswami CP, Nalamasu R, Li L, Jones D, Wei R, Liu W, Sarasani V, Janga SC and Chalasani N: Relationship between differential hepatic microRNA expression and decreased hepatic cytochrome P450 3A activity in cirrhosis. PLoS One. 8:e744712013. View Article : Google Scholar : PubMed/NCBI
21	Blaya D, Coll M, Rodrigo-Torres D, Vila-Casadesús M, Altamirano J, Llopis M, Graupera I, Perea L, Aguilar-Bravo B, Díaz A, et al: Integrative microRNA profiling in alcoholic hepatitis reveals a role for microRNA-182 in liver injury and inflammation. Gut. 65:1535–1545. 2016. View Article : Google Scholar : PubMed/NCBI
22	Matsuura K, De Giorgi V, Schechterly C, Wang RY, Farci P, Tanaka Y and Alter HJ: Circulating let-7 levels in plasma and extracellular vesicles correlate with hepatic fibrosis progression in chronic hepatitis C. Hepatology. 64:732–745. 2016. View Article : Google Scholar : PubMed/NCBI
23	Zhang J, Huang J, Liu W, Ding L, Cheng D and Xiao H: Identification of common oncogenic genes and pathways both in osteosarcoma and Ewing's sarcoma using bioinformatics analysis. J Immunol Res. 2022:36559082022.PubMed/NCBI
24	Wang Z, Zhang J, Feng T, Zhang D, Pan Y, Liu X, Xu J, Qiao X, Cui W and Dong L: Construction of lncRNA-Mediated competing endogenous RNA networks correlated With T2 asthma. Front Genet. 13:8724992022. View Article : Google Scholar : PubMed/NCBI
25	Zhong T, Li Z, You ZH, Nie R and Zhao H: Predicting miRNA-disease associations based on graph random propagation network and attention network. Brief Bioinform. 23:bbab5892022. View Article : Google Scholar : PubMed/NCBI
26	Li G, Sun J, Zhang J, Lv Y, Liu D, Zhu X, Qi L, Chen Z, Ye Z, Su X and Li L: Identification of Inflammation-related biomarkers in diabetes of the exocrine pancreas with the use of weighted gene Co-Expression network analysis. Front Endocrinol (Lausanne). 13:8398652022. View Article : Google Scholar : PubMed/NCBI
27	Mostafavi S and Morris Q: Combining many interaction networks to predict gene function and analyze gene lists. Proteomics. 12:1687–1696. 2012. View Article : Google Scholar : PubMed/NCBI
28	Wang S, Shen L and Luo H: Identification and Validation of Key miRNAs and a microRNA-mRNA Regulatory Network Associated with Ulcerative Colitis. DNA Cell Biol. 40:147–156. 2021. View Article : Google Scholar
29	Matsuura K, De Giorgi V, Schechterly C, Wang RY, Farci P, Tanaka Y and Alter HJ: Circulating let-7 levels in plasma and extracellular vesicles correlate with hepatic fibrosis progression in chronic hepatitis C. Hepatology. 64:732–745. 2016. View Article : Google Scholar : PubMed/NCBI
30	Xia S, Wang Z, Chen L, Zhou Y, Li Y, Wang S, Chen A, Xu X, Shao J, Zhang Z, et al: Dihydroartemisinin regulates lipid droplet metabolism in hepatic stellate cells by inhibiting lncRNA-H19-induced AMPK signal. Biochem Pharmacol. 192:1147302021. View Article : Google Scholar : PubMed/NCBI
31	Cutroneo KR, White SL, Phan SH and Ehrlich HP: Therapies for bleomycin induced lung fibrosis through regulation of TGF-beta1 induced collagen gene expression. J Cell Physiol. 211:585–589. 2007. View Article : Google Scholar : PubMed/NCBI
32	Kurtz CL, Fannin EE, Toth CL, Pearson DS, Vickers KC and Sethupathy P: Inhibition of miR-29 has a significant lipid-lowering benefit through suppression of lipogenic programs in liver. Sci Rep. 5:129112015. View Article : Google Scholar : PubMed/NCBI
33	Apte RS, Chen DS and Ferrara N: VEGF in signaling and disease: Beyond discovery and development. Cell. 176:1248–1264. 2019. View Article : Google Scholar : PubMed/NCBI
34	Aoki M and Fujishita T: Oncogenic Roles of the PI3K/AKT/mTOR Axis. Curr Top Microbiol Immunol. 407:153–189. 2017.PubMed/NCBI
35	Song YM, Lee YH, Kim JW, Ham DS, Kang ES, Cha BS, Lee HC and Lee BW: Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway. Autophagy. 11:46–59. 2015. View Article : Google Scholar :
36	Muthusamy A, Lin CM, Shanmugam S, Lindner HM, Abcouwer SF and Antonetti DA: Ischemia-reperfusion injury induces occludin phosphorylation/ubiquitination and retinal vascular permeability in a VEGFR-2-dependent manner. J Cereb Blood Flow Metab. 34:522–531. 2014. View Article : Google Scholar : PubMed/NCBI
37	Ash D, Sudhahar V, Youn SW, Okur MN, Das A, O'Bryan JP, McMenamin M, Hou Y, Kaplan JH, Fukai T and Ushio-Fukai M: The P-type ATPase transporter ATP7A promotes angiogenesis by limiting autophagic degradation of VEGFR2. Nat Commun. 12:30912021. View Article : Google Scholar : PubMed/NCBI
38	Hunt NJ, Kang SWS, Lockwood GP, Le Couteur DG and Cogger VC: Hallmarks of Aging in the Liver. Comput Struct Biotechnol J. 17:1151–1161. 2019. View Article : Google Scholar : PubMed/NCBI
39	Brenner C, Galluzzi L, Kepp O and Kroemer G: Decoding cell death signals in liver inflammation. J Hepatol. 59:583–594. 2013. View Article : Google Scholar : PubMed/NCBI
40	Efferth T: From ancient herb to modern drug: Artemisia annua and artemisinin for cancer therapy. Semin Cancer Biol. 46:65–83. 2017. View Article : Google Scholar : PubMed/NCBI
41	Li Q, Ma Q, Cheng J, Zhou X, Pu W, Zhong X and Guo X: Dihydroartemisinin as a sensitizing agent in cancer therapies. Onco Targets Ther. 14:2563–2573. 2021. View Article : Google Scholar : PubMed/NCBI
42	Hong DS, Kang YK, Borad M, Sachdev J, Ejadi S, Lim HY, Brenner AJ, Park K, Lee JL, Kim TY, et al: Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours. Br J Cancer. 122:1630–1637. 2020. View Article : Google Scholar : PubMed/NCBI
43	Zhang L, Liao Y and Tang L: MicroRNA-34 family: A potential tumor suppressor and therapeutic candidate in cancer. J Exp Clin Cancer Res. 38:532019. View Article : Google Scholar : PubMed/NCBI
44	van Rooij E and Kauppinen S: Development of microRNA therapeutics is coming of age. EMBO Mol Med. 6:851–864. 2014. View Article : Google Scholar : PubMed/NCBI
45	Teratani T, Tomita K, Furuhashi H, Sugihara N, Higashiyama M, Nishikawa M, Irie R, Takajo T, Wada A, Horiuchi K, et al: Lipoprotein Lipase Up-regulation in hepatic stellate cells exacerbates liver fibrosis in nonalcoholic steatohepatitis in mice. Hepatol Commun. 3:1098–1112. 2019. View Article : Google Scholar : PubMed/NCBI
46	Duan X, Meng Q, Wang C, Liu Z, Liu Q, Sun H, Sun P, Yang X, Huo X, Peng J and Liu K: Calycosin attenuates triglyceride accumulation and hepatic fibrosis in murine model of non-alcoholic steatohepatitis via activating farnesoid X receptor. Phytomedicine. 25:83–92. 2017. View Article : Google Scholar : PubMed/NCBI
47	Hu C and Jiang X: Role of NRP-1 in VEGF-VEGFR2-Independent Tumorigenesis. Target Oncol. 11:501–505. 2016. View Article : Google Scholar : PubMed/NCBI
48	Simons M, Gordon E and Claesson-Welsh L: Mechanisms and regulation of endothelial VEGF receptor signalling. Nat Rev Mol Cell Biol. 17:611–625. 2016. View Article : Google Scholar : PubMed/NCBI
49	Yang YL, Wang FS, Lin HY and Huang YH: Exogenous therapeutics of Microrna-29a attenuates development of hepatic fibrosis in cholestatic animal model through regulation of phosphoinositide 3-Kinase p85 Alpha. Int J Mol Sci. 21:36362020. View Article : Google Scholar : PubMed/NCBI
50	Mazo DF, de Oliveira MG, Pereira IV, Cogliati B, Stefano JT, de Souza GF, Rabelo F, Lima FR, Ferreira Alves VA, Carrilho FJ and de Oliveira CP: S-nitroso-N-acetylcysteine attenuates liver fibrosis in experimental nonalcoholic steatohepatitis. Drug Des Devel Ther. 7:553–563. 2013.PubMed/NCBI
51	Samali A, Cai J, Zhivotovsky B, Jones DP and Orrenius S: Presence of a pre-apoptotic complex of pro-caspase-3, HSP60 and HSP10 in the mitochondrial fraction of Jurkat cells. EMBO. 18:2040–2048. 1999. View Article : Google Scholar
52	Caruso Bavisotto C, Alberti G, Vitale AM, Paladino L, Campanella C, Rappa F, Gorska M, Conway de Macario E, Cappello F, Macario AJL and Marino Gammazza A: HSP60 Post-translational modifications: Functional and pathological consequences. Front Mol Biosci. 7:952020. View Article : Google Scholar : PubMed/NCBI
53	Macario AJ and de Macario EC: Molecular mechanisms in chaperonopathies: Clues to understanding the histopathological abnormalities and developing novel therapies. J Pathol. 250:9–18. 2020. View Article : Google Scholar