Effect of Compound Phyllanthus urinaria Ⅱ on Growth and Expression of miR-122 and IGF-1R of Hepatocellular Carcinoma Hep3B Cells in Nude Mice / 中国实验方剂学杂志

ObjectiveTo investigate the inhibitory effects and mechanism of the compound Phyllanthus urinaria Ⅱ （CPU Ⅱ）on the growth of transplanted hepatocellular carcinoma Hep3B2.1-7 （Short for Hep3R） cells in nude mice. MethodAfter the establishment of a xenograft model of hepatocellular carcinoma Hep3B cells in mice， the model mice were randomly divided into a model group， a high-dose CPU Ⅱ group （57.5 g·kg-1）， a low-dose CPU Ⅱ group （28.75 g·kg-1）， and a 5-fluorouracil （5-FU） group （0.025 g·kg-1）， with eight mice in each group. The mice in the high- and low-dose CPU Ⅱ groups were treated with drugs by gavage， once per day， and those in the model group were treated with the same volume of normal saline. The mice in the 5-FU group were treated by 5-FU by intraperitoneal injection， once every other day. After 28 days of administration， mice were sacrificed， and transplanted tumors were collected. Immunohistochemistry （IHC） was used to detect the expression of proliferating cell nuclear antigen （PCNA） of tumor tissues. Terminal-deoxynucleotidyl transferase-mediated nick end labeling （TUNEL） was used to detect cell apoptosis of tumor tissues. The mRNA expression of miR-122 and insulin-like growth factor 1 receptor （IGF-1R） in tumor tissues was detected by Real-time quantitative PCR （Real-time PCR）. The protein expression of CCAAT/enhancer-binding protein α （C/EBPα）， hepatocyte nuclear factor-4α （HNF-4α）， and IGF-1R in tumor tissues was detected by Western blot. ResultThe tumor suppression rates of the high- and low-dose CPU Ⅱ groups and the 5-FU group were 74.90%， 63.62%， and 64.15%， respectively. Compared with the model group， the CPU Ⅱ groups and the 5-FU group showed reduced weight （P<0.01） and volume of tumors （P<0.01）， decreased PCNA positive cells， shallow staining， increased apoptosis cells of transplanted tumor tissues （P<0.05， P<0.01）， increased expression of mRNA expression of miR-122 （P<0.01）， down-regulated mRNA expression of IGF-1R （P<0.01）， and up-regulated protein expression of C/EBPα and HNF-4α in nude mouse transplanted tumor tissues （P<0.01）. The expression of IGF-1R protein in the high-dose CPU Ⅱ group was down-regulated （P<0.05）. Compared with the low-dose CPU Ⅱ group， the high-dose CPU Ⅱ group showed increased apoptotic cells （P<0.01）， up-regulated mRNA expression of miR-122 （P<0.01）， and increased expression of C/EBPα and HNF-4α proteins （P<0.01）. ConclusionCPU Ⅱ has an obvious inhibitory effect on the growth of transplanted hepatocellular carcinoma Hep3B cells in nude mice. The mechanism of action is related to enhancing the expression of transcription factors HNF-4α and C/EBPα， thereby promoting the expression of miR-122 and inhibiting the expression of its target gene IGF-1R.

Effect of Compound Phylanthus urinaria Ⅱ in Regulating Expression of lncRNA CCAT1 in Hepatocellular Carcinoma HepG2 Cells / 中国实验方剂学杂志

Objective:To explore the anti-hepatoma effect of compound <italic>Phylanthus urinaria</italic> Ⅱ （ CPU Ⅱ） by inhibiting the expression of the long non-coding RNA （lncRNA） colon cancer associated transcript-1 （CCAT1） and restoring the expression of microRNA let-7a. Method:Real-time fluorescence quantitative polymerase chain reaction （PCR） was used to detect the expression of lncRNA CCAT1 in normal liver cells （LO2 cells） and hepatocellular carcinoma HepG2 cells， and the differences in expression between these two types of cells were compared. The methylthiazolyl tetrazolium（MTT） assay was used to detect the proliferation of HepG2 cells after treatment with different concentrations of CPU Ⅱ and 5-fluorouracil（5-FU） for 24， 48 and 72 h. Hepatocellular carcinoma HepG2 cells were cultured <italic>in vitro </italic>and set into three gropes： cell control group， CPU Ⅱ low-dose group （0.8 g·L^-1） and high-dose group （1.6 g·L^-1）. Real-time PCR was used to detect the mRNA expression of lncRNA CCAT1， microRNA let-7a and its target genes high mobility group protein A2（HMGA2）， and N-RAS in each grope. Western blot was used to detect the protein expression of HMGA2， and Cyclin D₁ in each grope. Result:As compared with LO2 cells， expression of lncRNA CCAT1 in HepG2 cells was significantly up-regulated （<italic>P</italic><0.05）. Results of MTT assay showed that the 50% inhibiting concentration（IC₅₀）of CPU Ⅱ and 5-FU on hepatocellular carcinoma HepG2 cells was 1.649， 0.044 648 g·L^-1respectively. As compared with the control group， CPU Ⅱ high-and low-dose groups （1.6， 0.8 g·L^-1） significantly inhibited the proliferation of HepG2 cells （<italic>P</italic><0.05）， and the effect was most remarkable in CPU Ⅱ high-dose group （<italic>P</italic><0.05）. The results of Real-time PCR showed that as compared with control group， the expression of lncRNA CCAT1 mRNA was significantly inhibited in CPU Ⅱ high-and low-dose groups （<italic>P</italic><0.05）， and the expression of microRNA let-7a mRNA was obviously up-regulated in high-dose group （<italic>P</italic><0.05）， but the expression of HMGA2 mRNA in CPU Ⅱ high-and low-dose groups as well as the expression of N-RAS mRNA in CPU Ⅱ low-dose group were down-regulated （<italic>P</italic><0.05）. Western blot results showed that as compared with the cell control group， the protein expression of HMGA2 and Cyclin D₁ in CPU Ⅱ high-and low-dose groups （1.6， 0.8 g·L^-1） was significantly down-regulated （<italic>P</italic><0.05）. Conclusion:CPU Ⅱ can inhibit the expression of lncRNA CCAT1， recover the expression of microRNA let-7a， and suppress the mRNA and protein expression of related downstream target genes in hepatoma cells line HepG2， thereby inhibiting the proliferation of hepatocellular carcinoma cells and exerting anti-hepatocellular carcinoma effect.

Effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on Proliferation, Apoptosis and Autophagy of Hepatocellular Carcinoma Huh7 Cells / 中国实验方剂学杂志

Objective:To observe the effect and investigate the mechanism of compound Phyllanthus urinaria Ⅱ(CPU Ⅱ)on proliferation, apoptosis and autophagy of human hepatoma cell line Huh7. Method:Huh7 cells were cultured in vitro and divided into blank control group, high-dose CPU Ⅱ group (40 g·L-1),low-dose CPU Ⅱ group (20 g·L-1), and 5-FU group (0.04 g·L-1). Methye thiazolye telrazlium(MTT) assay was used to detect the inhibitory effect of CPU Ⅱ on proliferation of human hepatoma Huh7 cells. The apoptosis rate was observed by Annexin V-FITC/PI flow cytometry; the changes of autophagosomes in each group were observed by monodansylcadaverin (MDC) staining; Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of phosphatidylinositol 3-kinase (PI3K), Serine-threonine protein kinase 2(Akt2), B-cell lympoma-2(Bcl-2) and Microtubule-associated protein 1 light chian 3(LC3Ⅱ) and Western blot was used to detect the protein expression of Akt2 and LC3Ⅱ. Result:CPU Ⅱ(40, 20 g·L-1) significantly inhibited the hepatoma cell line Huh7 and induced apoptosis, with an apoptosis rate of 51.72% and 19.74% respectively, significantly higher than that of control group (PPPPConclusion:CPUⅡ had obvious inhibitory effect on the proliferation of hepatoma cell line Huh7, and the mechanism may be related to inhibiting the activation of PI3K/Akt signaling pathway to induce apoptosis and autophagy.

Experimental Study on In-vivo Effect of Compound Phyllanthus urinaria Ⅱ on Duck Hepatitis B Virus / 广州中医药大学学报

Objective To evaluate the in-vivo effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on duck hepatitis B virus(DHBV).Methods Thirty ducks with congenital infection of DHBV,which were DHBV-DNA positive confirmed by polymerase chain reaction(PCR),were randomly divided into 5 groups: DHBV control group,high-,middle-,and low-dosage CPU Ⅱgroups(in the dose of 33.6,18.6 and 8.4 g-1?d-1,respectively),and Lamivudine group(20 mg?kg-1?d-1).The serum DHBV-DNA level of all ducks was detected by quantity real-time fluorescence PCR before and after medication.Results On the 7th,14th,21st,and 28th day of medication and on the 5th day of suspension of medication,the serum DHBV-DNA level in both of the high-and middle-dosage CPU Ⅱ groups was obviously lower than that before medication and than that in the DHBV control group(P