Mechanism of Si Junzitang in Inhibiting Hepatocellular Carcinoma Progression Through Regulating O-GlcNAcylation of NF-κB p65 in Paracancerous Tissues / 中国实验方剂学杂志

ObjectiveTo investigate the molecular mechanism by which Si Junzitang in intervening in the development of hepatocellular carcinoma （HCC） by regulating the O-linked β-N-acetylglucosamine modification （O-GlcNAcylation） of nuclear factor kappa-B （NF-κB） p65 in the paracancerous tissues. MethodThe orthotopic liver cancer mouse model was established. Twenty-four C57BL/6 mice were randomly divided into four groups： Normal group， model group， Si Junzitang low-dose group （10 g·kg-1）， and Si Junzitang high-dose group （25 g·kg-1）， with 6 mice in each group. The O-GlcNAcylation level and phosphorylation modification level of p65 in the paracancerous tissues were detected using Western blot. The O-GlcNAcylation of p65 was assessed using immunoprecipitation （IP）. The mRNA expression of interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， transforming growth factor-β1 （TGF-β1）， vascular endothelial growth factor A （VEGFA）， matrix metalloproteinase-2 （MMP-2）， matrix metalloproteinase-9 （MMP-9） in the paracancerous tissues was detected using real-time quantitative polymerase chain reaction （Real-time PCR）. The tumor number， liver weight， locomotor activity， grip strength， and Qi status of the mice were observed and analyzed. ResultCompared with the normal group， the model group showed a significant decrease in O-GlcNAcylation in the paracancerous tissues （P<0.01）， a significant decrease in p65 O-GlcNAcylation （P<0.01）， a significant increase in p65 phosphorylation （P<0.01）， significantly elevated mRNA levels of cytokines IL-6， TNF-α， TGF-β1， VEGFA， MMP-2， and MMP-9 （P<0.01）， significantly increased liver weight （P<0.01）， significantly declined grip strength， number of grid crossings， and number of vertical stand-ups （P<0.01）， and significantly dwindled Qi status （P<0.01）. Compared with model group， the Si Junzitang low-dose and high-dose groups showed significantly increased levels of O-GlcNAcylation in the paracancerous tissues （P<0.05， P<0.01）， significantly upregulated p65 O-GlcNAcylation levels （P<0.05， P<0.01）， and significantly decreased p65 phosphorylation levels （P<0.01）. In the Si Junzitang low-dose group， the mRNA levels of IL-6， TGF-β1， and VEGFA significantly decreased （P<0.05， P<0.01）. In the Si Junzitang high-dose group， the mRNA levels of IL-6， TNF-α， TGF-β1， VEGFA， MMP-2， and MMP-9 significantly decreased （P<0.01）， the number of tumors larger than 3 mm in diameter significantly decreased （P<0.01）， and liver weight significantly decreased （P<0.05）. Additionally， grip strength， number of grid crossings， and number of vertical stand-ups significantly increased （P<0.05， P<0.01）， along with a significant increase in qi status （P<0.01）. ConclusionSi Junzitang can inhibit the progression of orthotopic HCC in mice， which may be achieved by increasing the O-GlcNAcylation level in the paracancerous tissues， enhancing the O-GlcNAcylation of p65， inhibiting the phosphorylation modification of p65， and ultimately suppressing the expression of downstream IL-6， TNF-α， TGF-β1， VEGFA， MMP-2， and MMP-9.

Injurious Effect of Cisplatin on the Function of Hypothalamus-pituitary-adrenal/gonadal Axis in Mice and the Intervention Effect of Dehydroepiandrosterone / 实验动物与比较医学

ObjectiveTo study the pathway of cis-dichlorodiamineplatinum (DDP) inhibiting the synthesis of steroid hormones in mice, and to observe the intervention effect of dehydroepiandrosterone (DHEA).MethodsSixty adult ICR mice were randomly divided into three groups: control group, DDP modeling group, and DHEA group, with 10 male and 10 female mice in each group. The DDP modeling group mice were intraperitoneally injected with DDP solution at a dose of 2.5 mg·kg-1·d-1, once every 3 days, a total of 7 times. On the same day of modeling, the control group mice were injected with an equal amount of physiological saline intraperitoneally. The DEHA treatment group mice were treated with DDP and given a dose of 8.3 mg·kg-1·d -1 of DHEA by gavage for 21 consecutive days. The changes of fatigue indexes of mice were observed by open field, grip and rod rotation tests. The morphology changes of adrenal gland, testicular and ovarian tissue were observed by pathological section and HE staining. The levels of serum steroid hormones were detected by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The mRNA and protein expression levels of the related genes of the hypothalamus, hypophysis, adrenal, testis and ovary were tested by real-time fluorescent quantitative PCR (RT-qPCR) and Western blotting.ResultsCompared with control group, both male and female mice in DDP modeling group were significantly losing weight (P＜0.05), their abilities in horizontal movement and vertical movement decreased (all P＜0.05), and the stay time and grip also significantly decreased (all P＜0.05) in female mice. Indexes of fatigue were improved after DHEA supplement (all P＜0.05). In the DDP modeling group, the arrangement of spermatogenic cells at all levels in the testicular tissue was disordered and the testicular interstitial edema was observed, and a large number of primordial follicles in the ovarian tissue were activated, the number of atresia follicles increased, and the number of granulosa cells in the follicles decreased; while in the DHEA group, the damaged phenotype of testicles and ovaries was significantly improved. Compared with control group, the levels of serum testosterone and dihydrotestosterone in both male and female DDP modeling mice significantly decreased (P＜0.01), the pregnenolone was down-regulated but corticosterone was up-regulated significantly (P＜0.05) in male mice, the corticosterone was down-regulated significantly (P＜0.05) in female mice. Compared with the DDP group, after DHEA supplement, the pregnenolone in male mice and the progesterone in female mice increased significantly (P＜0.05), but the pregnenolone in female mice and the progesterone in male mice decreased significantly (P＜0.05). Compared with control group, the expression levels of Cyp21a1 and Cyp11a1 genes in the adrenal gland and Gnrh gene in the hypothalamus of male and female mice in the DDP modeling group significantly decreased (all P＜0.05); the expression levels of Hsd3b2 gene in the adrenal gland, Star, Cyp11a1, and Lhr genes in the ovaries, Crh, Pomc, and Lhb genes in the hypothalamus, pituitary, and pituitary of female mice significantly decreased (all P＜0.05); the expression levels of Star gene and StAR protein in the testicles of male mice, as well as Fshb and Lhb genes in the pituitary gland, were significantly down-regulated (all P＜0.05). After DHEA supplement, compared with the DDP modeling group, the mRNA expression levels of Cyp17a1 in the adrenal gland of male mice and Cyp17a1, Lhr and Fshr genes in testis were down-regulated significantly (P＜0.05); the expression level of Cyp11a1 gene in the adrenal gland of female mice was also decreased (P＜0.05); while the expression levels of Hsd3b2 gene in the adrenal gland, Star, Cyp11a1, Hsd3b2 and Lhr gene in the ovary, and Lhb gene in the pituitary gland were all up-regulated ( P＜0.05).ConclusionThe function of hypothalamus-pituitary-adrenal/gonadal axis was inhibited by DDP intermittent injection, especially in female. Supplementation of DHEA can help regulate the homeostasis of steroid hormone levels.

Transcriptome profile and its partly verification of human hepatocellular carcinoma cells exposed to Yuzhizi () seed extract.

OBJECTIVE: To investigate the efficacy of Yuzhizi seed extract (FAQSE) on inhibiting the proliferation of hepatocellular carcinoma (HCC) cells in vitro and to explore the anti-HCC action mechanism of FAQSE. METHODS: Human HCC HepG2 and Huh7 cells were used to investigate the anti-HCC effect of FAQSE. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method was used to measure cell viability. Affymetrix microarray was adopted to detect the expression of transcriptome. The differentially expressed genes (DEGs) of each cell line were identified. For co-DEGs of both cell lines, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were enriched using the Database for Annotation, Visualization and Integrated Discovery (DAVID), and the network analysis of protein-protein interaction (PPI) was mapped using the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape software. Some important genes in the PPI network of co-DEGs were selected to verify by quantitative real-time reverse transcription-polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. RESULTS: FAQSE decreased the viability of HepG2 and Huh7 cells. There were 211 co-upregulated and 86 co-downregualted genes in both cell lines after FAQSE treatment. The enriched GO terms of co-upregulated DEGs were primarily involved cell-cell adhesion, viral process, transcription initiation from RNA polymerase II promoter, positive regulation of transcription from RNA polymerase II promoter and actin cytoskeleton organization. The GO terms of co-downregulated DEGs were mainly enriched in the processes of SRP-dependent cotranslational protein targeting to membrane, viral transcription, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, translational initiation and rRNA processing. Main KEGG pathways of co-upregulated DEGs were endocytosis, glutathione metabolism, protein processing in endoplasmic reticulum, synaptic vesicle cycle and lysosome. The major KEGG pathways of co-downregulated DEGs were ribosome, biosynthesis of amino acids, arginine and proline metabolism, systemic lupus erythematosus and complement and coagulation cascades. The top 10 co-DEGs with high hub nodes in STRING analysis were ribosomal protein S27a, transferrin, ribosomal protein S20, ribosomal protein L9, protein phosphatase 2 regulatory subunit B alpha, transthyretin, thioredoxin reductase 1, ribosomal protein L3, ribophorin I and ribosomal protein L24. Alpha-fetoprotein (AFP) was also co-downregulated and contained in the PPI network. The mRNA and protein expression of most verified genes was consistent with the results of co-DEGs analysis. And the AFP level was significantly reduced after FAQSE treatment. CONCLUSIONS: A series of genes and pathways of HepG2 and Huh7 cells were changed after FAQSE treatment, which might be the targets of FAQSE against HCC and worthy of further study. AFP might be important one of them.

Da Chaihutang Inhibits Hepatocellular Carcinoma by Regulating p38 MAPK/IL-6/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveThis study aims to investigate the efficacy and underlying mechanism of Da Chaihutang （DCHT） in treating hepatocellular carcinoma （HCC） in vitro and in vivo. MethodWe employed methyl thiazolyl tetrazolium （MTT） assay and crystal violet staining to observe the proliferation of Hepa1-6 liver cancer cells treated with DCHT at different doses （0， 125， 250， 500， 1 000 mg·L-1） for different time periods （1， 2， 4， 8 days）. The orthotopic liver cancer model was established by injection of 1×106 Hepa1-6 cells into mouse， and then the model mice were randomly assigned into six groups： blank， model， DCHT （0.21， 0.625， 1.875 g·kg-1， ig， qd）， and positive control （5-fluorouracil， 25 mg·kg-1， ip， qod）. After 14 days of administration， the mice were sacrificed， and the liver samples were collected and fixed in 4% paraformaldehyde for hematoxylin-eosin （HE） staining. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， Cytoscape 3.7.2， STRING， and DAVID were used for the searching of the key targets of DCHT in treating HCC， the construction of protein-protein interaction （PPI） network， and the Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment. Quantitative real-time PCR was performed to determine the mRNA level of interleukin-6 （IL-6） in Hepa1-6 cells and liver tissue. Western blotting was employed to measure the protein levels of the proteins involved in the mitogen-activated protein kinase （MAPK） and signal transducers and activators of transcription 3 （STAT3） signaling pathways. ResultDCHT （500， 1 000 mg·L-1） treatment for 4 and 8 days inhibited the proliferation of Hepa1-6 cells in a dose- and time-dependent manner （P<0.05）. The in vivo assay showed that DCHT （high dose， 1.875 g·kg-1） treatment for 14 days led to high differentiation and unobvious heterogeneity of HCC cells and small necrotic area compared with the model group. Network pharmacology analysis predicted that the potential targets of DCHT in the treatment of HCC were mainly the inflammation cytokines such as IL-6， interleukin-1β （IL-1β）， and tumor necrosis factor-alpha （TNF-α） in HCC microenvironment. The potential signaling pathways involved in the treatment were mainly associated with HCC growth and differentiation， including MAPK and STAT3 signaling pathways. Compared with the blank group， DCHT （1 000 mg·L-1） treatment for 1， 2， 4， and 8 days down-regulated the mRNA level of IL-6 in Hepa1-6 cells （P<0.05）. Similar results were observed in the livers of mice treated with DCHT （0.625， 1.875 g·kg-1）. The in vitro assay demonstrated that DCHT （1 000 mg·L-1） treatment for 4 and 8 days and DCHT （500， 1 000 mg·L-1） treatment inhibited the phosphorylation of extracellular signal-regulated kinases 1/2 （ERK1/2）， c-Jun NH2-terminal kinase/stress-activated protein kinase （JNK）， p38 MAPK， and STAT3 in a dose- and time-dependent manner （P<0.05）. The in vivo assay showed that DCHT （0.625 and 1.875 g·kg-1） treatment only inhibited the phosphorylation of p38 MAPK and STAT3 （P<0.05）. ConclusionThe present study indicates that DCHT can inhibit liver cancer cell proliferation by regulating p38 MAPK/IL-6/STAT3 signaling pathway.

Application and evaluation of moral education materials of scientific research cases in the teaching of Experimental Traditional Chinese Medicine / 中华医学教育探索杂志

Objective:To evaluate the teaching effect of moral education materials implied in scientific research cases in the teaching of "Experimental Traditional Chinese Medicine (TCM)".Methods:The moral education materials implied in scientific research cases of "Experimental TCM" were extracted and skillfully integrated into the teaching of professional knowledge. A questionnaire survey was conducted with questions as "whether it is good to demonstrate the potential humanistic spirit by case teaching, whether this helps improve your interest in science and whether this teaching form affects your study, life and work attitude in the future" "Ten specific items from scientists' moving deeds that touch students and their recognitions" to assess the teaching effect.Results:95.8 percent of students affirmed this teaching form and thought it helped improve their interest in scientific exploration. 87.5 percent of students considered the humanistic spirit would affect their study, life and work in the future. 77.1-89.6 percent of students held a positive attitude to the ten items derived from the scientists' moving stories. Among these items, the percentages of the two items, "the spirit of being able to endure loneliness, work hard to make contributions selflessly without seeking for rewards or reputations" and "having respect forpeople and their scientific research achievements with courage to challenge the authorities of scientific research" are the lowest and highest, respectively.Conclusion:It is good to apply the moral education materials implied in the scientific research cases in the teaching of "Experimental TCM", which basically achieves the teaching goal, but there is still room for improvement.

Practice and reflections on the teaching reform of Experimental Traditional Chinese Medicine guided by scientific research cases / 中华医学教育探索杂志

Experimental Traditional Chinese Medicine is an emerging discipline that plays an important role in cultivating innovative talents of traditional Chinese medicine (TCM). In recent years, with the rapid development of TCM and the new requirements of positioning, and also combined with the college students' cognitive characteristics, we have revised and republished the "Experimental TCM" (Third Edition) textbook, which focuses on introducing knowledge by adopting relevant scientific research cases. This test book was used in the teaching of undergraduates of batch 2013 in the eight-year program in Shanghai University of Traditional Chinese Medicine. After-class questionnaires showed that this teaching mode, guided by scientific research case, is not only helpful for students to develop their quality and ability of adopting modern experimental methods initially in the study and development of TCM, but also able to spread the great achievements of TCM researches. The teaching mode is also conducive to enhancing students' sense of responsibility for the modernization of TCM. Therefore, it is suggested that the course of Experimental TCM should be promoted in the colleges and universities of TCM.