The Ethyl Acetate Fraction Of Hedyotis Diffusa Willd Induces Apoptosis In Human Hepatocellular Carcinoma Hep3B Cells Via The JNK/Nur77 Pathway

Objective:Hepatocellular carcinoma(HCC)is one of the malignant tumors with strong heterogeneity and high mortality,and its morbidity and mortality are increasing year by year.The options for HCC treatment are limited.Since symptoms in the early stage are not obvious,most patients have already been diagnosed in the middle and advanced stages,opportunities for surgical treatment are then lost.Medicines are the main treatment for middle and advanced liver cancer.Traditional Chinese medicines have great potential in the application of HCC due to their unique advantages.Hedyotis diffusa Willd(HDW)is a whole herb of the genus Hedyotis(Rubiaceae),which has been used as injection to improve the quality of life of patients with HCC.In searching for effective anti-HCC agents from HDW,our preliminary drug screening found that the ethyl acetate fraction of HDW(EHDW)displayed the strongest anti-HCC activitity.However,the molecular mechanisms have not been defined.In this study,we aim to evaluate the anti-HCC activity of EHDW against Hep3B cells in vitro and in vivo,as well as explore the underlying mechanisms.Methods:(1)The zebrafish toxicity of different fractions from HDW was peformed by immersion administration.(2)The in vivo anti-HCC effect of different HDW fractions was evaluated on a zebrafish tumor model.(3)HE staining and Hoechst 33258 staining were used to analyze EHDW-induced apoptosis in zebrafish.(4)MTT assay was used to evaluate the anti-proliferation activity of EHDW against Hep3B cells.(5)Apoptosis induced by EHDW was analyzed by Hoechst 33258 staining and Annexin V-FITC/PI staining.(6)The expression of apoptosis-related proteins such as Bcl-2,Bax,cyto c,cleaved-caspase 3,PARP,cleaved-PARP were detected by Western blotting.(7)Western blotting was used to detect the levels of JNK and Nur77.The role of JNK/Nur77 pathway in EHDW-induced mitochondrial apoptosis was investigated with SP600125.(8)The main chemical components of EHDW were identified by HPLC-QTOF-MS.Results:(1)The toxicity of the extracts gradually increased with the decrease of the polarity of solvents.(2)EHDW showed the strongest tumor cell growth inhibitory effect on zebrafish tumor model.(3)EHDW induced apoptosis of Hep3B cells in zebrafish.(4)EHDW inhibited the proliferation of Hep3B cells in vitro.(5)EHDW induced changes in nuclear morphology and apoptotic rate of Hep3B cells.(6)EHDW up-regulated the expression of Bax/Bcl-2 ratio,cyto c,caspase-3 and PARP.(7)EHDW up-regulated the level of p-JNK(Thr183/Tyr185)and p-Nur77(Ser351).SP600125 inhibited JNK phosphorylation,which in turn inhibited its downstream Nur77.The blockage of JNK by SP600125 reversed EHDW-induced JNK/Nur77 pathway and mitochondrial apoptosis.(8)A total of 55 compounds were identified by EHDW,including iridoids,anthraquinones,flavonoids.Conclusion:EHDW induced mitochondrial apoptosis in Hep3B cells by activating JNK/Nur77 signaling pathway,suggesting that EHDW may be a potential therapeutic drug for HCC.