Mechanism of Biejiajian Wan Against EMT of Hepatocellular Carcinoma Cells Through NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Biejiajian Wan （BJJW） on transforming growth factor-β1 （TGF-β1）-induced epithelial-mesenchymal transition （EMT） of HepG2 cells， and explore its mechanism against EMT of hepatocellular carcinoma cells. MethodHepG2 cells were randomly divided into a blank group， a TGF-β1 model group （10 μg·L-1 TGF-β1）， a low-dose BJJW group （10 μg·L-1 TGF-β1+0.55 g·kg-1 BJJW）， a medium-dose BJJW group （10 μg·L-1 TGF-β1+1.1 g·kg-1 BJJW）， a high-dose BJJW group （10 μg·L-1 TGF-β1+2.2 g·kg-1 BJJW）， and a sorafenib group （10 μg·L-1 TGF-β1+0.03 g·kg-1 sorafenib）. The EMT model was induced by 10 μg·L-1 TGF-β1 in HepG2 cells. After treatment with corresponding medicated serum， cell counting kit -8 （CCK-8） assay was used to detect cell proliferation. Cell migration ability was detected by the Transwell assay and wound healing assay. The protein expression related to EMT and nuclear factor-kappa B （NF-κB） signaling pathway was detected by cell immunofluorescence assay and Western blot. ResultCompared with the blank group 4 days later， the TGF-β1 model group showed fusiform and loose cells with widened gap and antennae reaching out， decreased protein expression of E-cadherin （P<0.05）， and increased protein expression of N-cadherin and vimentin （P<0.05）， which indicated that the EMT model was properly induced in HepG2 cells by TGF-β1 stimulation for 4 days. After 48 hours of treatment with the corresponding medicated serum， each medication group showed inhibited proliferation of HepG2 cells that had undergone EMT， especially the low- and high-dose BJJW groups （P<0.01）， and the medium-dose BJJW group showed increased E-cadherin protein expression （P<0.05） and decreased p-p65， N-cadherin， and vimentin protein expression （P<0.05）， as compared with the TGF-β1 model group. As revealed by the transwell assay and wound healing assay， TGF-β1 enhanced the migration ability of HepG2 cells （P<0.05， P<0.01） compared with the results in the blank group， compared with the TGF-β1 model group， the medication groups showed inhibited migration ability of HepG2 cells （P<0.05， P<0.01）. Compared with the blank group， the TGF-β1 model group promoted the expression of p65 and Snail into the nucleus. Compared with the TGF-β1 model group， the medication groups inhibited the expression of p65 and Snail into the nucleus. ConclusionBJJW may inhibit the EMT， proliferation， and migration of HepG2 cells induced by TGF-β1 by suppressing the NF-κB signaling pathway to exert an anti-hepatocellular carcinoma effect.

Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/ β-catenin Pathway / 中国实验方剂学杂志

Objective:To study the effect of Biejiajian Wan on the epithelial-mesenchymal transition （EMT） of rat hepatic oval cells induced by transforming growth factor- β1（TGF-β1）， in order to explore its mechanism in reversing EMT. Method:WB-F344 cells were divided into five groups： blank group， TGF-β1 model group （10 μg·L-1TGF-β1）， low-dose group （10 μg·L-1TGF-β1+0.55 g·kg-1Biejiajian Wan）， medium-dose group （10 μg·L-1TGF-β1+1.1 g·kg-1Biejiajian Wan）， high-dose group （10 μg·L-1TGF-β1+2.2 g·kg-1Biejiajian Wan）. Except blank group， TGF-β1 was used to induce WB-F344 cells in all of the remaining groups to construct an EMT model. After the cells were treated with low， medium and high doses of Biejiajian Wan serum， the changes of migration ability of WB-F344 cells were detected by cell scratching test. The expressions of E-cadherin， N-cadherin and Vimentin were detected by Western blot. Real-time PCR was used to detect the changes in the expression of β-catenin mRNA. The expression of β-catenin was detected by cell immunofluorescence assay. Result:Compared with normal WB-F344 cells， the intercellular space of WB-F344 cells became loose from tight， and the morphology changed from cobblestone to fibroblast after TGF-β1 induced WB-F344 cells for 4 days， and the expression of E-cadherin protein decreased， while the expression of N-cadherin protein increased （P<0.01）， indicating that the EMT model of WB-F344 cells was successfully built. Compared with the blank group， the migration ability of WB-F344 cells in TGF-β1 model group was enhanced （P<0.01）， compared with TGF-β1 model group， Biejiajian Wan could significantly inhibit the migration ability of WB-F344 cells； specifically， the low-dose group had no statistical significance， and the medium and high-dose groups had statistical significance （P<0.05）. Western blot results showed that compared with the blank group， the expression of E-cadherin decreased， whereas those of N-cadherin and Vimentin increased in the TGF-β1 model group （P<0.01）， compared with TGF-β1 model group， E-cadherin protein expression was increased in the low， medium and high-dose groups， while the expressions of N-cadherin and Vimentin was decreased； specifically， the low-dose groups had no statistical significance， and the medium and high dose groups had statistical significance （P<0.05，P<0.01）. Real-time PCR results showed that compared with the blank group， the mRNA expression of β-catenin in the TGF-β1 model group was increased （P<0.05）， whereas compared with TGF-β1 model group， the mRNA expression of β-catenin in the low， medium and high-dose groups of Biejiajian Wan was reduced （P<0.01）. The results of cellular immunofluorescence showed that compared with the blank group， the fluorescence expression of β-catenin in the cell nucleus was enhanced in the TGF-β1 model group； and compared with the TGF-β1 model group， the expression of β -catenin in the cell nucleus of the low， medium and high-dose groups of Biejiajian Wan decreased， and the inhibitory effect of Biejiajian Wan on β-catenin in the cell nucleus was positively correlated with its concentration. Conclusion:Biejiajian Wan may reverse the EMT process that TGF-β1 induced WB-F344 cells， and inhibit the migration of WB-F344 cells by inhibiting Wnt/β-catenin signaling pathway.

Mechanism of Biejia Jianwan in Suppressing Epithelial-mesenchymal Transition in DEN-induced Rat Hepatocellular Carcinoma via TGF-β/Smad Pathway / 中国实验方剂学杂志

Objective:To study the effect of Biejia Jianwan on expressions of signal molecules and target genes of transforming growth factor-β (TGF-β)/Smad pathway in diethylnitrosamine (DEN)-induced rat hepatocellular carcinoma, and explore the mechanisms of Biejia Jianwan suppressing the invasion of hepatocellular carcinoma. Method:The rats were divided into three group, namely normal group, model group and Biejia Jianwan group (2.2 g·kg-1·d-1). Rats in Biejia Jianwan group and model group received intraperitoneal injections of DEN to induce sequential chronic inflammation, cirrhosis and hepatocellular carcinoma. At the sign of cirrhosis, rats in Biejia Jianwan group began taking Biejia Jianwan by gavage for 6 weeks. Rat blood was collected to measure serum levels of biochemical markers of liver function tests, including alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL), albumin(Alb), γ-glutamyl transpeptadase(GGT), alkaline phosphatase(ALP). Rat livers were fixed in formalin and stained with hematoxylin-eosin (HE)staining， quantitative real-time PCR was used to test the mRNA expressions of TGF-β1， and Western blot was used to test protein expressions of TGF-β1, Smad2/3, p-Smad2/3, N-cadherin, E-cadherin and Vimentin. Result:All of the levels of biochemical markers showed no difference in Biejia Jianwan group and model group. Biejia Jianwan could improve the pathological changes of balloon-like degeneration, edema, and necrosis in liver cancer tissues. Importantly, the treatment dramatically decreased the mRNA expression of TGF-β1(P<0.01), and the protein expressions of TGF-β1, p-Smad2(P<0.01). Besides, the protein expression of N-cadherin and Vimentin were decreased significantly (P<0.01). Conclusion:Biejia Jianwan can inhibit epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells activated via TGF-β/Smad pathway by reducing TGF-β1 expression, so as to suppress the metastasis and invasion of hepatocellular carcinoma.

Research of differences of miRNAs expression profiles in different metastatic potential HCC cell lines / 中国药理学通报

Aim To screen the differential microRNA (miRNA) expression profiles of different metastatic po-tential liver cancer cell lines,and predict miRNAs-reg-ulated target genes and their functions. Methods To-tal RNA was extracted and the miRNA expression pro-files were obtained by miRNA microarray chip hybrid-ization. The miRNAs whose expression had significant difference were selected by analyzing the miRNA difference expression profiles of the two different meta-static potential liver cancer cell lines, namely MHCC-97H(high-metastasis) and Hep3B(non-metastasis), which were compared with normal hepatocytes L02 re-spectively. Moreover, we analyzed the miRNA differ-ential expression profile between liver cancer cell lines MHCC-97H and Hep3B. The miRNAs were verified by qPCR and target genes were predicted by four softwares (TargetScan, miRanda, miRWalk, miRDB). To un-derstand the biological functions of predicted target genes, bioinformatics analysis was performed. Results The miRNA microarray results showed that the ex-pression of miR-192-5p and miR-215-5p significantly increased in liver cancer cell lines (MHCC-97H, Hep3B) when compared with normal hepatocytes L02, while miR-130a-3p and miR-196a-5p were significantly reduced; compared with Hep3B, the expression of miR-224-5p markedly increased in liver cancer cell line MHCC-97H, while miR-146a-5p, miR-483-3p and miR-200b-3p were significantly reduced. The re-sults of qRT-PCR were consistent with chip results. Conclusion There are differences of miRNA expres-sion profiles in different metastatic potential liver canc-er cell lines MHCC-97H, Hep3B, and they may par-ticipate in regulating the development and invasion of hepatocellular carcinoma.

Research progress of miRNAs targeting GSK-3β in regulation of hepatocellular carcinoma invasion and metastasis / 南方医科大学学报

Invasion and metastasis are key factors contributing to the high mortality rate of patients with hepatocellular carcinoma (HCC) involving a complex mechanism. In the invasion and metastasis of HCC, miRNAs can serve as either oncogenes or tumor suppressor genes to regulate the differentiation and proliferation of tumor cells being and play important roles in tumorigenesis, angiogenesis, invasion and metastasis. This review summarizes the recent progress in research of the molecular mechanisms by which miRNAs targeting GSK-3β regulate HCC invasion and metastasis and examines the roles of miRNAs in hepatocellular carcinoma cell proliferation, apoptosis, invasion, metastasis, and GSK-3β regulation.

Inhibitory effect of Biejiajian pills on HepG2 cell xenograft growth and expression of β-catenin and Tbx3 in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the molecular mechanism by which Biejiajian pills inhibit hepatocellular carcinoma in a nude mouse model bearing HepG2 cell xenograft.</p><p><b>METHODS</b>The inhibitory effect of Biejiajian pills on the growth of HepG2 cell xenograft in nude mice was observed. Immunohistochemical method was used to examine proliferating cell nuclear antigen (PCNA) expression in HepG2 cell xenograft, and TUNEL method was employed to detect the cell apoptosis; the expression levels of β-catenin and Tbx3 were measured by Western blotting.</p><p><b>RESULTS</b>Biejiajian pills significantly suppressed the growth of HepG2 cell xenograft in nude mice. The tumor-bearing mice treated with a high and a moderate dose of Biejiajian pills showed significantly increased apoptosis rate of the tumor cells [(22.9±1.220)% and (14.7±0.50)%, respectively] compared with the control group [(5.5±0.90)%, P<0.05]. Treatment with Biejiajian pills significantly decreased the expressions of PNCA, β-catenin, and Tbx3 in the cell xenograft (P<0.05).</p><p><b>CONCLUSIONS</b>Biejiajian pills can inhibit the growth of HepG2 cell xenograft in nude mice and promote tumor cell apoptosis possibly by inhibiting PNCA expression and the Wnt/β-catenin signaling pathway.</p>

Blejiajian pill inhibited the proliferation, adhesion, and invasion of hepatoma carcinoma cells: an experimental research / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate effects of biejiajian pill (BP) on the proliferation, adhesion, and invasion of hepatoma carcinoma cells (HepG2), and to primarily explore the mechanisms for fighting against metastasis and invasion.</p><p><b>METHODS</b>Using sero-pharmacological methods, HepG2 cells were respectively cultured by high and middle dose BP containing serums and the vehicle serum. Using MTT colorimetry, cell adhesion test, and Transwell invasion test, effects of BP on the proliferation, adhesion, and invasion of HepG2 cells were detected, thus further exploring the mechanisms for fighting against the metastasis and invasion of HepG2 cells.</p><p><b>RESULTS</b>High and middle dose BP containing serums could significantly prohibit the growth and proliferation, the adhesion and invasion of HepG2 cells on the basilar membrane. Besides, these effects were correlated with the concentrations of BP.</p><p><b>CONCLUSION</b>BP could effectively inhibit the growth and proliferation, adhesion and invasion of HepG2 cells.</p>

Proteomic analysis of chronic restraint stress-induced Gan (肝)-stagnancy syndrome in rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To analyze the proteomic characteristics of Gan (肝)-stagnancy syndrome (GSS) by seeking the differential protein in blood and tissues of GSS model rats.</p><p><b>METHODS</b>GSS model rats were established by chronic restraint stress, keeping rats in restrain chamber for 6 h every day for 21 successive days. Their blood and liver samples were collected at the end of experiment for differential protein detection with methods of isoelectrofocusing and polyacrylamide SDS-PAGE, silver staining, and scanning. The gel images were analyzed with Imagemaster 2D Elite software, and the excavated differential protein spots were identified with matrix assistant laser resolving TOF mass spectrometry, Western blot, ELISA, and RT-PCR, respectively.</p><p><b>RESULTS</b>A method for isolating the protein in blood serum and tissues by two-dimensional gel electrophoresis was established and optimized. Six serum proteins and three liver proteins that differentially expressed were identified. The down-regulated differential proteins in serum of GSS model rats were serum albumin precursor, beta 1 globin, antibody against muscle acetylcholine receptor, Ig lambda-2 C region, and transthyretin (TTR), and those in liver tissue were aryl sulfotransferase, enoyl-CoA hydratase, and TTR. TTR down-regulation was found in both serum and liver. Preliminary biological information analysis showed that these differential proteins involved in immune, neuroendocrine, nutrition, and substance metabolism.</p><p><b>CONCLUSION</b>Proteomic analysis of differential proteins showed that TTR, aryl sulfotransferase, and enoyl-CoA hydratase expressions are downregulated in the GSS model rats, suggesting that the susceptibility of cancer could be enhanced by chronic stress.</p>

Effect of Baoganning on serum and hepatic leptin and its receptor levels in rats with liver fibrosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanism underlying the protective effects of a traditional Chinese medicinal formula, Baoganning, against liver fibrosis.</p><p><b>METHODS</b>Male Wistar rats were subjected to injection of carbon tetrachloride- peanut oil mixture and given daily 5% alcoholic beverage, and 2 days after the injection, Baoganning was administered intragastrically at two different doses for 6 weeks. Radioimmunoassay was used to detect serum leptin level, and immunohistochemistry employed to examine the effect of Baoganning on expressions of leptin and its receptor in the liver tissue of the rats.</p><p><b>RESULTS</b>Compared with the normal control group, the rats in the liver fibrosis model group and Baoganning-treated groups showed significantly increased serum leptin levels (P<0.01), and the serum leptin level was significantly lower in Baoganning group than in the liver fibrosis model group (P<0.01). Baoganning significantly reduced the hepatic expression of leptin and OB-Rb in rats with liver fibrosis in comparison with their expression in the model group (P<0.01).</p><p><b>CONCLUSION</b>Baoganning can effectively ameliorate liver fibrosis in rats possibly through reducing serum leptin level and inhibiting hepatic leptin and its receptor expressions.</p>