Methyl helicterte ameliorates liver fibrosis by regulating miR-21-mediated ERK and TGF-β1/Smads pathways
Authors: Xing Lin
Min He
Jinlan Nie
Renbin Huang
Facheng Bai
Yuanyuan Wei
Xiaolin Zhang
Quanfang Huang
Jinbin Wei
Zhongpeng Lu
Shujuan Wen
Journal: International Immunopharmacology (2019)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: ERK pathway; Hepatic fibrosis; Hepatic stellate cells; Methyl Helicterte; MicroRNA-21 (miR-21)
Affiliations: The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China., Guangxi Medical University, Nanning 530021, China., Department of Biochemistry, University of Arkansas Medical School, 4301 W. Markham, Little Rock, AR 72205-7199, USA., Guangxi Medical University, Nanning 530021, China. Electronic address: .

Abstract:  Methyl helicterate (MH) has been reported to have protective effects against CCl4-induced hepatic injury and fibrosis in rats, but its protective mechanism, especially on hepatic stallete cells (HSCs), remains unclear. Recently, our pilot experiment showed that MH could inhibit miR-21 expression in HSC-T6 cells, suggesting that miR-21 may be one of the targets of MH to intervene liver fibrosis. To verify the hypothesis, the present study would focus on the regulatory effect of MH on the miR-21-mediated ERK and TGF-β1/Smads pathways. Briefly, rats were intraperitoneally injected with 0.5 ml porcine serum (PS) twice a week for 24 weeks to induce liver fibrosis, and meanwhile, the rats were treated with MH from weeks 16 to 24. In vitro experiment, miR-21 expression in HSC-T6 cells was up- or down-regulated using lentiviral transfection assay. Collagen accumulation, inflammatory cytokines, cell apoptosis, miR-21 expression, and activation of the ERK and TGF-β1/smad2/3 pathways were then assessed. The results showed that MH treatment markedly alleviated PS-induced liver injury, as evidenced by the attenuation of histopathological changes and the decrease in serum alanine and aspartate aminotransferases activity. MH significantly decreased the content of inflammatory cytokines and recruited the anti-oxidative defense system. Moreover, MH treatment significantly decreased miR-21 expression and inhibited the activation of the ERK and TGF-β1/smad2/3 pathways in liver tissues. In vitro experiments showed that MH strongly inhibited HSC-T6 cell activation and reduced collagen accumulation. Interestingly, miR-21 overexpression significantly promoted HSC-T6 cell proliferation, reduced HSC apoptosis, and increased collagenation, while these abnormal changes induced by miR-21overexpression were significantly reversed by MH treatment. Furthermore, miR-21 overexpression notably activated the ERK and TGF-β1/Smads pathways via repressing SPRY2 and Smad7 expression respectively, however, these effects were largely abolished by MH treatment. In conclusion, our study demonstrates that MH significantly alleviates PS-induced liver injury and fibrosis by inhibiting miR-21-mediated ERK and TGF-β1/Smads pathways. 

Retracted: No

PubPeer comments:
 Fig 1: Fig 1A was corrected, but they did not correct B ![file] , 
 B6: ![file] , 
 Fig 2C: ![file] ![file] , 
 Fig 4C: ![file] ![file] , 
 Fig 5F: ![file] ![file] , 
 Fig 6E: Flip lane 5 horizontally. ![file] ![file] , 
 Fig 7F: ![file] ![file] , 
 Fig 8F: Flip lane 6 vertically ![file] ![file] , 
 #2 Fig 1B3. ![file] , 
 #8 Fig 8F again: ![file] , 
 Fig 6D6.  (D) HSC-T6 cell apoptosis was detected by flow cytometry analysis; D1 to D6 represented the normal control group, lentivirus-NC group, lentivirus-up group, 3, 6 and **12 u03bcmol l MH-treated** groups, respectively . ![file] What happened here? , 
 Fig. 1.  MH ameliorated PS-induced liver injury. (A) The appearance of the liver samples. A1 to A6 represented the normal control group, MH control group, **PS model group, 16.72, 33.45** and 66.90 mg kg MH-treated groups, respectively . ![file] Fig. 3 from [Wen et al (2019)] .  MHHA ameliorated alcohol-induced liver injury. (A) Histological changes were observed using H E staining (200u00d7); A1: normal control, A2: MHHA control, **A3: alcohol-model group**, A4-A6: MHHA-treated groups (**16.72, 33.45** and 66.90 mg kg) . ![] Three pairs of liver images are the same. Was the fibrosis (untreated, or MH-treated) the result of porcine serum or alcohol? , 
 For the sake of completeness, Figs 5D and 7D. ![file] , 
 Below: Fig 7F.  MH significantly inhibited the TGF-u03b21 Smads and ERK pathways after miR-21 overexpression in HSC-T6 cells....(F) ...the phosphorylation of p-Smad2 3 and p-ERK were detected by Western blotting; band 1 to 6 represented the normal control group, lentivirus-NC group, lentivirus-up group, 3, 6 and 12 u03bcmol l MH-treated groups, respectively . Above: Fig 4 from [Wei et al (2019)] .  The expression of Colu2010I ... was detected by immunohistochemical staining (u00d7400) and Western blot analysis. u201c1u201d to u201c6u201d represent the normal control group, 20 ng mL PDGFu2010BB group, 100 u03bcM PD98059 group, 3.75, 7.5 or 15 u03bcM MHu2010treated groups, respectively . ![] Another sighting! Fic 7C from [Wen et al (2019)] .  MHHA inhibited HSC proliferation and activation. ... (C) Protein expression of u03b1-SMA was determined with Western blot assay. Band 1u20136 represented the normal group, TGF-u03b21 group, LY364947 group, and MHHA treatment groups (3, 6 and 12 u03bcmol L). ![file] , 
 #12 The rest of Fig 6: ![file] For comparison, Fig 8B from [Wen et al (2019)] :  MHHA significantly induced HSC apoptosis. (A-B) Cell apoptosis was assessed by AO EB staining (200u00d7) and flow cytometry, respectively; the arrow indicates hepatocyte apoptosis. A1 to A6 (or B1 to B6) represented the normal group, TGF-u03b21 group, LY364947 group, and MHHA treatment groups (3, 6 and 12 u03bcmol L), respectively. ![file] , 
 Corrigendum [February 2020] . -------------------------------------------------- The authors regret that the wrong Fig. 1 (A3 to A5) was published. The correct figures are provided in this corrigendum. The authors would like to apologize for any inconvenience caused. ![] ----------------------------------- This is the correction mentioned by Paxillus Obscurosporus in comment #1, which left all the other problems in place. ,