Study On The Effects Of Celastrol On Hepcidin And Its Upstream Proteins Expressions In IL-6-Stimulated HepG2 Cells

Background and Objective Celastrol has significant anti-inflammatory,anti-immune and anti-cancer effects,and has therapeutic effects on a variety of diseases.However,its toxicity（especially the hepatorenal toxicity）has limited its extensive clinical application.Elucidation of the molecular mechanisms of celastrol toxicity can better promote its clinical transformation.The investigations by us and others suggested that celastrol might affect iron metabolism.It is well known that iron excess produces toxicity,especially hepatorenal toxicity.Therefore,this work use human hepatocellular carcinoma cell line（HepG2）cells as model,to study the effects on hepcidin（the main regulator on iron balance in vivo,the decrease of its expression can lead to the increase of intestinal iron absorption,leading to the increase of iron content in body and causing liver and kidney toxicity and other damages）and its upstream proteins.This work could give preliminary answer to the question whether celastrol have the possibility to influence iron balance,thus providing one new sight for exploring the toxicity mechanism and detoxification strategy for celastrol.Methods Different concentrations of celastrol were added into HepG2 cells for different time,and the cytotoxic effect of celastrol was detected by CCK-8.The optimal safe dose of celastrol was screened out by calculating IC₅₀.The hepcidin-induced cell model was created by stimulation of HepG2 with recombinant inflammatory cytokine IL-6.The expressions of intracellular phosphorylated NF-κB,total NF-κB,IL-6 and hepcidin were detected by Western blot,to verify the success of the model induction.The effects of celastrol on hepcidin and its up streaming modulators in IL-6 stimulated HepG2 cells were determined by real-time fluorescent quantitative PCR and western blot.The upstream modulators included are BMP 6（BMP6）,transferrin receptor 2（TFR2）,iron element regulatory proteins（HJV）,the hemochromatosis（HFE）protein,the expression of interleukin 6（IL-6）,with the activation of NF-κB（p-NF-κB）level being used as the index to verify the celastrol effectiveness in the experiment system.Finally,si RNA interference technology was used to knockdown BMP6,TFR2 and HFE to establish gene modified cell models,and the alterations of the effects of IL-6 stimulation and celastrol treatment in these gene-modified model cells were observed,so as to further clarify the roles of these upstream regulatory proteins in IL-6’s and celastrol’s affecting hepcidin.Results The survival rates of HepG2 cells decreased gradually with the increase of celastrol concentration and treatment time.Based on toxicity experiment,the celastrol dose were set to 1.2μmol/L in the following study to minimize the toxicity.The unstimulated HepG2 cells constitutively expressed hepcidin and its upstream regulators,which could not be inhibited by celastrol,though the well-known effects of celastrol,i.e.,inhibiting p-NF-κB were revealed.In non-starved cells,IL-6（50 ng/ml）for 12 h could dramatically increase the expressions of the proteins and m RNAs of hepcidin and its upstream proteins in HepG2 cells（P<0.05）.Celastrol could significantly lowered cytokine IL-6-sitmulated elevations of hepcidin and its upstream regulators.Down-regulation of the protein expressions of TFR2,or BMP6 or HFE by si RNA decreased the protein and m RNA expressions of hepcidin in HepG2cells,as well as affect the levels of other upstream proteins of hepcidin.The effects of IL-6and celastrol in regulating hepcidin are reduced in cells with TFR2 or BMP6 being knocked down.Conclusion Our results for the first time found that celastrol could inhibit the elevations of hepcidin in IL-6 stimulated HepG2 cells,with the up-streaming BMP6,TFR2and HFE being possible involved.The findings suggested that celastrol be a potential natural compound to increase iron content in vivo,hence it deserves further study whether increasing iron concentration in the body be involved in causing liver and kidney toxicity by this compound.