RESUMEN

Liver fibrosis is a critical health issue in the world due to its rapidly increasing prevalence. It is of great demand to develop effective drugs for the treatment of liver fibrosis. 5-methoxytryptophan (5-MTP) has been reported to play an important role in anti-inflammatory, anti-cancer, myocardial-protective effects. However, the anti-fibrotic effect of 5-MTP is never covered in liver. Here, we investigated anti-fibrotic effects of 5-MTP on liver fibrosis and its underlying mechanism. In vitro, 5-MTP treatment could inhibit TGF-ß1-induced elevated levels of collagen I, collagen III, fibronectin and α-smooth muscle actin (SMA) by stimulating autophagy process. Mechanically, the expression of FOXO3a was enhanced by 5-MTP and then repressed the level of miR-21, eventually leading to a restoration of autophagy-related gene ATG5. Furthermore, rescue experiments showed 5-MTP could activate autophagy process and suppress the activation of LX-2 cells by regulating FOXO3a/miR-21/ATG5 pathway. Consistently, 5-MTP significantly attenuated CCl4-induced hepatic fibrosis in rat model. In conclusion, our research discovered that 5-MTP effectively alleviated liver fibrosis in vitro and in vivo, which provided new insights into the application of 5-MTP for liver fibrosis.

Asunto(s)

Proteína 5 Relacionada con la Autofagia/biosíntesis , Autofagia/efectos de los fármacos , Proteína Forkhead Box O3/biosíntesis , Cirrosis Hepática/metabolismo , MicroARNs/biosíntesis , Triptófano/análogos & derivados , Animales , Autofagia/fisiología , Proteína 5 Relacionada con la Autofagia/genética , Tetracloruro de Carbono/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Proteína Forkhead Box O3/genética , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/tratamiento farmacológico , Masculino , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Triptófano/farmacología , Triptófano/uso terapéutico

RESUMEN

OBJECTIVE: To construct the hepatitis B virus (HBV) X gene recombinant and to induce the expression of X protein. METHODS: HBV DNA was extracted from the serum of patient with hepatitis B. The X gene was amplified by PCR using the primers with EcoRI and HindIII digestion sites, and then cloned into pronucleus expression vector pMAL-C2X, which was detected by EcoRI and HindIII digestion and sequence. Finally, the recombinant was induced by IPTG to express X protein in JM109. RESULTS: The band similar to X gene was amplified by PCR. There were fragments similar to X gene when the recombinant was digested by the enzyme digestion. It was tested by DNA sequence that the correct and entire opening reading frame of HBV X gene was inserted. The X protein was expressed by the IPTG induction. CONCLUSION: Pronucleus expression recombinant pMAL-C2X-HBV-X is constructed successfully and with the IPTG induction, the recombinant pMAL-C2X-HBV-X can express the X protein in E. coli JM109, which lays the foundation for the HBV X protein purification and its biological study.

Asunto(s)

Virus de la Hepatitis B/genética , Transactivadores/biosíntesis , Transfección , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Análisis de Secuencia de ADN , Transactivadores/genética , Proteínas Reguladoras y Accesorias Virales

RESUMEN

OBJECTIVE: To clarify the nature of human hepatocyte DNA synthetic stimulated factor (HDSSF) and to lay a foundation for its gene cloning and expression. METHODS: We amplified the target gene fragment by degenerating PCR, labelled the fragment as a probe to screen human fetal liver cDNA library, and then analyzed the HDSSF gene sequence. RESULTS: A 600 base pair fragment was obtained and labelled successfully with DIG. Human HDSSF sequence was determined by screening the library and gene sequencing. Its cDNA chain was composed of 748 base pairs including 594 base pair open read-code frame and 5' and 3' terminal noncode district sequence. CONCLUSION: By screening cDNA library, the HDSSF gene cloning has been gained successfully. HDSSF should be a new hepatocyte growth substance which consists of 198 amino acid residues.

Asunto(s)

ADN Complementario/genética , Factor de Crecimiento de Hepatocito/genética , Clonación Molecular , Humanos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN

RESUMEN

OBJECTIVE: To construct the eukaryotic expression recombining vector on human The pGEM-hepatocyte DNA synthetic stimulated factor (hHDSSF) with gene cloning. METHODS: hHDSSF, a mid-recombining vector, was constructed by T-A cloning. After restriction endonuclease Not I digestion, the target fragment was subcloned into eukaryotic vector pcDNA3. 1hisB to construct the eukaryotic expression recombinant pcDNA3. 1hisB-HDSSF. RESULTS: The forward insert recombinant pcDNA3. 1hisB-HDSSF was screened and obtained with restriction endonuclease Kpn I digestion and it was detected by DNA sequence analysis. CONCLUSION: The eukaryotic expression recombinant pcDNA3. 1hisB-hHDSSF on hHDSSF is constructed successfully, which lays a foundation for building a stable eukaryotic expression cell strain and expressing hHDSSF.

Asunto(s)

Factor de Crecimiento de Hepatocito/biosíntesis , Clonación Molecular , Células Eucariotas/metabolismo , Expresión Génica , Vectores Genéticos , Factor de Crecimiento de Hepatocito/genética , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética