Effect of PEP-1-SOD1 on Cell Apoptosis in SAP Rats / 胃肠病学

Background: Severe acute pancreatitis (SAP) is characterized by diffuse pancreatic hemorrhage and tissue necrosis with high mortality. PEP-1-SOD1 is a fusion protein synthesized by genetic engineering technology. It has a high stability and certain anti-inflammatory effects. Aims: To investigate the effect of PEP-1-SOD1 on cell apoptosis in SAP rats. Methods: A total of 24 male Wistar rats were divided into control group, SAP group and experimental group. SAP rat model was established by infusion of 5% sodium taurocholate. Thirty minutes before the establishment, rats in experimental group were abdominal subcutaneously injected with 8.0 mg/kg PEP-1-SOD1, and rats in SAP group were injected with same dose of 0.9% NaCl solution. Histopathological score of pancreatic tissue were evaluated; apoptosis of pancreatic acinar cell was determined by TUNEL. The mRNA and protein expressions of caspase-3 were detected by fluorescent quantitative PCR and Western blotting, respectively. Results: After 24 hours of model establishment, serum amylase and lipase, mRNA and protein expressions of caspase-3 in SAP group and experimental group were significantly higher than those in the control group (P<0.05), however, serum amylase and lipase in experimental group were significantly lower than those in SAP group (P<0.05), while mRNA and protein expressions of caspase-3 were significantly increased (P<0.05). After 6, 24 hours of model establishment, histopathological score, apoptotic index in SAP group and experimental group were significantly higher than those in the control group (P<0.05), however, histopathological score in experimental group was significantly lower than that in SAP group (P<0.05), while apoptotic index was significantly increased (P<0.05). Conclusions: PEP-1-SOD1 may increase the apoptosis of pancreatic acinar cells through regulating the expression of apoptosis related gene caspase-3 in SAP rats, thereby reducing the pathological damage of pancreatic tissue and promoting the recovery of pancreatic function.

Inhibitory effect of PEP-1-mediated recombinant hepatocyte nuclear factor 4 alpha transduction on hepatocellular carcinoma cells / 第二军医大学学报

Objective To investigate cell penetrating peptide (PEP-1)-mediated transduction of recombinant hepatocyte nuclear factor 4 alpha (HNF4α) protein into hepatocellular carcinoma (HCC) cells, and to observe the effect of the fusion protein P-HNF4α on HCC cells. Methods The expression vector pET28a-P-HNF4α was constructed. The prokaryotic expression condition of fusion protein P-HNF4α was optimized. Recombinant P-HNF4α carrying cell penetrating peptide PEP-1 was obtained by abundant expression, purified by affinity chromatography, and was concentrated and dialyzed. P-HNF4α was transduced into HCC cells. The transduction efficiency was analyzed by Western blotting analysis. Sub-cellular localization of P-HNF4α was detected by Western blotting analysis with nuclear and cytoplasmic extracts and confirmed by immunofluorescence assay. Real-time RT-PCR was used to examine the gene expression of HCC cells. The proliferation of HCC cells was detected with CCK-8 kit. The migration and invasion of HCC cells were detected by wound-healing assay and trans-well invasion assay, respectively. Results P-HNF4α was efficiently transduced into Huh7 cells and located in the nucleus as mediated by PEP-1. P-HNF4α significantly up-regulated the expression of characteristic hepatocyte markers and down-regulated the "stemness" genes in Huh7 cells (P<0.05 or P<0.01). Moreover, the proliferation (P<0.05), migration (P<0.001) and invasion (P<0.05) of HCC cells were significantly suppressed by fusion protein P-HNF4α. Conclusion P-HNF4α can induce the differentiation of HCC cells to mature hepatocytes and reduce the malignancy phenotype of HCC cells, suggesting that PEP-1-mediated HNF4α protein transduction may be a potential strategy for HCC differentiation therapy.

Chlorogenic Acid Improves Neuroprotective Effect of PEP-1-Ribosomal Protein S3 Against Ischemic Insult

Chlorogenic acid (CGA) possesses various biological activities such as anti-oxidant, anti-inflammatory, and anti-diabetic activities. In the present study, we examined the effect of CGA on the transduction efficiency of PEP-1-ribosomal protein S3 (PEP-1-rpS3) into cells and brain tissues, and its neuroprotective potential against ischemia/reperfusion. We found that, in the presence of CGA, the transduction efficiency of PEP-1-rpS3 into astrocytes and the CA1 region of the hippocampus was enhanced, compared to its transduction in the absence of CGA. Also, cell viability data demonstrated that the sample treated with CGA + PEP-1-rpS3 exhibited improved cell viability against hydrogen peroxide (H2O2)-induced toxicity more significantly than the sample treated with PEP-1-rpS3 alone. Also, in a gerbil ischemia model, data demonstrated that following the ischemic insult, the group treated with PEP-1-rpS3 + CGA showed markedly enhanced protection of neuron cells in CA1 region of hippocampus, compared to those treated with CGA or PEP-1-rpS3 alone. Taken together, these results suggest that CGA may improve the transduction efficiency of protein transduction domain (PTD) fusion proteins into target cells or tissues, thereby enhancing their therapeutic potential against various diseases.

Study on the Penetrability of PEP-1-P27mt for Cell Membranes in Vitro / 华中科技大学学报（医学）（英德文版）

Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, prokaryotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS transformed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentrations in 2 h. PEP-1-P27mt of 1 μmol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological protocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.

Cell-penetrating peptide PEP-1 mediated transmembrane delivery of enhanced green fluorescent protein in vivo of mouse / 基础医学与临床

Objective To investigate the in vivo transduction capability of fusion protein PEP-1-EGFP with mice.Methods Two prokaryotic expression plasmids pET15b-EGFP and pET15b-PEP-1-EGFP were constructed and transformed into E.coli BL21(DE3) to express EGFP and fusion protein PEP-1-EGFP,respectively.The expressed EGFP and PEP-1-EGFP were purified with Ni2+-resin affinity chromatography.Five hundred micrograms of EGFP and PEP-1-EGFP fusion protein were injected into mouse through caudal vein,respectively,the mice were euthanized and perfused with PBS 2 hours after administration.Then,the heart,brain,liver,spleen and kidney were removed and sectioned with a cryostat at 7 ?m for visualization with a inverted fluorescent microscope.ResultsThe brain,heart,liver,spleen and kidney injected with PEP-1-EGFP showed bright and homogenous green fluorescence whereas that with EGFP showed no green fluorescence at all.Conclusion The successful expression and purification of PEP-1-EGFP fusion protein and its efficient transduction into mice in vivo provide a basis for the research on transmembrane delivery of macromolecule drugs mediated by the cell-penetrating peptide,PEP-1.