ABSTRACT
ObjectiveTo explore the mechanism of Huangqisan （HQS） in regulating autophagy to alleviate hepatic steatosis and improve non-alcoholic fatty liver disease （NAFLD） based on adenosine 5'-monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway. MethodThe main chemical components and targets of HQS and NAFLD-related targets were collected from database and the intersection targets were used for Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. The protein-protein interaction （PPI） network was constructed， and in vivo experimental verification was conducted. Sixty C57BL/6J male mice were randomly divided into normal control group （NCD）， model group high-fat diet （HFD）， metformin group （MET， 0.25 g·kg-1）， low-dose Huangqisan group （HQS-L， 0.5 g·kg-1）， and the high-dose Huangqisan group （HQS-H， 1 g·kg-1）， with 12 mice in each group after a one-week acclimatization period. NAFLD model was induced by HFD， and intragastric administration was performed at the same time， once a day for 13 weeks. Random blood glucose， serum total cholesterol （TC）， triglyceride （TG）， non-esterified fatty acid （NEFA）， low density lipoprotein-chdesterol （LDL-C） levels， and liver TG content were determined. The liver weight was weighed， and liver index was calculated. Hematoxylin-eosin （HE） staining， oil red O staining， transmission electron microscope （TEM）， real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and Western blot were used to verify the effect and reveal the potential mechanism of C57BL/6J mice in vivo. ResultThrough network pharmacology analysis， combined with previous studies， it was predicted that HQS may improve NAFLD by regulating autophagy via the AMPK/mTOR signaling pathway. The result of in vivo experiment showed that， as compared with NCD group， random blood glucose， body weight， serum TC， LDL-C， NEFA， liver weight， liver index， and liver TG content of mice in the HFD groups were significantly increased （P<0.01）. HE staining showed massive lipid droplets （LDs） vacuolated， oil red O staining showed lipid accumulation in liver cells， and no obvious autophagosomes and autolysosome were observed under TEM. The relative mRNA expression of LC3A、LC3B、AMPKα1 and protein expression of AMPK， phosphory phosphorylated（p）-AMPK， and p-AMPK/AMPK were significantly down-regulated （P<0.01）， while the protein expression of microtubule-associated protein 1 light chain 3 （LC3）Ⅱ/Ⅰ and p-mTOR was significantly up-regulated （P<0.01）. As compared with HFD groups， liver weight， serum TG， and NEFA levels in HQS-L and HQS-H groups were significantly deceased （P<0.05， P<0.01）. HE staining and oil red O staining showed the improvement of liver pathological changes after HQS administration. Under TEM， a small amount of autophagosome and autolysosome were observed. Besides， liver index was significantly decreased in the HQS-L group （P<0.01）， and random blood glucose， serum TC level and liver TG content were significantly decreased in the HQS-H group （P<0.05）. The results of Western blot and Real-time PCR showed that the mRNA expression of LC3A and LC3B and the protein expression of LC3Ⅱ/Ⅰ， p-AMPK， and p-AMPK/AMPK were significantly up-regulated （P<0.01）， while the mRNA expressions of p62 and protein expression of p62 and p-mTOR were significantly down-regulated （P<0.05， P<0.01）. ConclusionHQS may promote autophagy and restore autophagy flux via the AMPK/mTOR signaling pathway to alleviate hepatic steatosis improving NAFLD.
ABSTRACT
The standard surgery intern orientation is very important. The surgery intern orien-tation is completed by surgery education division (SED) in the U.S.A. The orientation includes speech from director of surgery department. the interaction part presided by surgical teachers, topic groups for discussing. Then the faculties of SED will explain the core competences which are required by accred-itation council on graduate medical education (ACGME), educational activities of department, the re-view process for resident physicians. The orientation also includes occupation development plan for residents; manners, teamwork and communication to patients; surgical basic skills training, system management, feedback and evaluation etc. The expectation of this article is for the improvement of Chinese surgery intern orientation.
ABSTRACT
Objective To investigate the effect of RNAi-mediated survivin gene with conditionally replicating adenovirus Ad-de-lE1b55KD-shRNA/survivin-EGFP silencing on propagation and apoptosis of colon carcinoma cell lines HT-29 .Methods Ad-de-lE1b55KD-shRNA/survivin-EGFP was transfected to transplantation tumor on athymic mouse ,replication defective adenovirus and liposome vector which was contained the same shRNA as Ad-delE1b55KD-shRNA/survivin-EGFP were as control one .Then the change of transplantation tumor on athymic mouse was tested ,and TUNEL was used to assay the apoptosis rate of transplantation tumor cells .Results After transfection of Ad-delE1b55KD-shRNA/survivin-EGFP ,the quality of transplantation tumor on athymic mouse were reduced and the apoptosis rate of transplantation tumor cells were higher than replication defective adenovirus and lipo -some vector which was contained the same shRNA (P<0 .05) .Conclusion The effect of Ad-delE1b55KD-shRNA/survivin-EGFP to colon carcinoma cell on propagation and apoptosis were higher than replication defective adenovirus and liposome vector .
ABSTRACT
Objective To construct the conditionally replicating adenovirus vector Ad-delE1b55kD-shRNA/Survivin-EGFP that can transfects into HT-29 effectually and selectively and contains shRNA targeting to human Survivin gene.Methods EGFP geoe and shRNA gene were inserted into pAd-delE1b55kD,then pAd-delE1b55kD-shRNA/Survivin-EGFP was obtained.The plasmid and pBHGE3 were cotransfected into HEK-293 cell to obtain the conditionally replicating adenovirus vector Ad-delE1b55kD-shRNA/Survivin-EGFP.Then DNA of the adenovirus vector was extracted and identified.The transfection efficiency of the vector to HT-29 and LO2 was detected.The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot.Results EGFP gene and shRNA gene targeting to human Survivin mRNA were inserted into conditionally replicating adenovirus vector successfully,proven by sequencing.The transfection efficiency to HT-29 of Ad-delE1b55kD-shRNA/Survivin-EGFP was significantly higher than replication defective adenovirus and liposome vector(P
ABSTRACT
Objective:To reseach the effects of focused ultrasound on the capability of proliferation that human hepatocarcinoma cell SMMC7721 have in vitro,and to determine the mechamism of this therapy initially.Methods:To irradiate the SMMC7721 cell which is in logarithmic growth time with ultrasound.The dosage of ultrasound was 0.8MHz 20W 25s,which make 70% of SMMC7721 cell that had been irradiated alive.The changes in cell morphology,biochemistry and gene expression were observed with immunohistochemistry and hybridization in situ.Results:The ultrastructure of SMMC7721 cell was changed by focused ultrasound,and the ultrasound could inhibit the expression of VEGF,PCNA,VEGF mRNA and PCNA mRNA( P
ABSTRACT
Objective: To construct a replication-competent adenovirus shuttle plasmids named pAd-delE1b55kD,which can transfect the cell of tumor targeted.Methods: delete the E1B55 region of pXC1 and retain a site for Bgl II by such technique of gene recombination as nested PCR;verify the correctness of pAd-delE1b55kD by gel image analysis and sequencing the gene order.Results: The 2279~3326 region of E1B in pXC1 was deleted.The 2024 basic radical C was mutated to T,the 2252 basic radical C to T and the 2261 basic radical G to T.Stop codon TGA?TAG and TAA were inserted.A site for Bgl II was retained in downstream of the stop codon.Conclusion: The replication-competent adenovirus shuttle plasmids named pAd-delE1b55kD is constructed,which provided a carrier for the gene therapy of tumor targeted.