| Hepatocellular carcinoma(HCC),as one of the malignant tumors with high morbidity and mortality,threatens human health seriously.The development of HDAC inhibitors(HDACI)showed attractive prospect in the treatment of a variety of cancers.Many innovative drugs of HDACI emerged in the treatment of malignant tumors with high efficiency and low toxicity.Therefore,computer virtual screening combined with activity testing in vitro were used to search for the compounds with significant HDAC inhibitory activity.Finally,we focused on the diarylheptanoids in Miq.The anti-proliferation activity test and the action mechanisms of the extractions and diarylheptanoids were studied in vitro.In addition,chemical liver injury is an important initial factor in the occurrence and development of various liver diseases,including HCC.Due to its strong antioxidant and free radical scavenging activity,we investigated for the first time about the protective effect of the extraction and medium polarity fraction of Alpinia oxyphylla on CCl4-induced liver injury in rats.Objective:Based on the clinical phenomenon,we focused on HCC and the target of HDAC.By the means of computer added virtual screening,we tried to look for the compounds from natural products database with potential HDAC inhibitory activity and the possible sources of traditional Chinese medicines.Then we investigated a certain kinds of compounds in one of the traditional Chinese medicines,hoping to find the lead compounds with significant activity and value for further research.In addition,this study described the effects and mechanisms of other activities that related to this herb.Methods:(1)By means of pharmacophore generation in computer aided drug design(CADD)technology,the virtual screening was carried out to screen compounds with good binding activity to HDAC from traditional Chinese medicine database of over 50 thousand natural products,and these compounds were analyzed and summed up in order to determine the possible sources of Chinese herb.(2)The MTT and lactate dehydrogenase(LDH)release assay were used to investigate the cytotoxicity of the extracts from traditional Chinese medicine on tumor cells.Hoechst 33342 staining,Annexin V/PI double staining,the generation of ROS and mitochondrial membrane potential(MMP)etc.were carried out to dectect the anti-proliferation and pro-apoptosis effects of Alpinia oxyphylla extract on HepG2 cells.(3)The protein expression of Bax/Bcl-2,Bad,p53,PARP,Cyt-c and Cleaved caspase-3/9,ERK,JNK,p38,AKT and Acetyl-Histone H3 were detected by Western Blot analysis.The preliminary mechanism of its anti-proliferation and pro-apoptosis were exploited.(4)Based on the CCl4-induced liver injury model in rats,we detected the hepatoprotective effect of Alpinia oxyphylla extract and the medium polarity fraction against hepatic injury,the liver function indexes such as AST,ALP,ALT were detected and the HE staining were used to take the microscopy observation.The liver biochemical index changes such as SOD,MDA,GSH were used to judge the activity and the mechanism in vivo.(5)The HPLC analytical method was applied for analysis the main components of the extractions of Alpinia oxyphylla.Results:(1)It was found that four compounds with potential HDAC inhibitory activity were found from four kinds of Chinese herbal medicines by the screening of the pharmacophore model.They are Turmeric,Fructus Alpiniae oxyphyllae,Common Cnidium Fruit and Oats(Fried).(2)MTT results showed that Alpinia oxyphylla and the petroleum ether extraction layer(PE),which containing diarylheptanoids could significantly inhibit the proliferation of several kinds of cancer cells in vitro.These effects may related to it’s potential HDAC inhibitory activity.As for these effects were less frequently reported in the literature,Alpinia oxyphylla was chosen as the research object preliminary.(3)Further results of activity test showed that PE showed a strong inhibition and pro-apoptotic effects on a variety of tumor cells,especially HepG2 cells,as well as inhibited the expression of Ace-Histone H3.(4)PE could significantly reduce the survival rate and promote the LDH release of HepG2 cells.PE significantly increased ROS generation and decreased MMP;PE also increased the ratio of Bax/Bcl-2 and the expression of Cleaved caspase-3/9,Bad,p53,PARP,as well as promoted Cyt-c release.At the same time,PE could promote the phosphorylation of JNK and p38,and inhibit the phosphorylation of ERK and AKT.(5)The diarylheptanoids in fructus Alpinia oxyphylla revealed that Yakuchinone A,Yakuchinone B and Oxyphyllacinol could significantly inhibit the proliferation of HepG2 cells at the concentration of 25-100 μg/mL with dose-dependent manner.Yakuchinone B also showed inhibitory effect on the expression of Ace-Histone H3.(6)The ethanol extract(CE)and dichloromethane layer of Alpinia oxyphylla extract(DE)could obviously improve the growth status and liver function indexes such as AST,ALP,ALT against CCl4-induced liver injury model in rats.These effects could be worked by improving liver intracellular SOD activity and GSH levels,as well as reducing MDA content.Conclusion:(1)It will be very helpful to screen and research a certain kinds of natural compounds quickly and accurately when combined with the activity oriented test and target oriented screening of computer aided drug design technologies.(2)It has been discovered for the first time that the PE has extensive cytotoxic on several tumor cells and significantly pro-apoptotic effects in HepG2 cells.The mechanisms may be related to the mitochondrial apoptosis pathway and the phosphorylation of ERK,JNK,p38 and AKT,as well as the HD AC inhibition.(3)CE and DE were discovered for the first time about the hepatoprotective effect against CCl4-induced hepatic injury in rats,and the possible mechanisms may be attributed to the free radical scavenging effect and inhibition of lipid peroxidation. |
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