The Application Methods of Mitogen-activated Protein Kinase Signal Pathway Inhibitors SP600125 and SB203580 in Long-term in Vivo Experiments / Métodos de aplicación de los inhibidores SP600125 y SB203580 de la vía de señalización de la proteína quinasa activada por mitógeno en experimentos in vivo a largo plazo

ABSTRACT Objective: To explore the application methods of mitogen-activated protein kinase signal pathway inhibitors SP600125 and SB203580 in long-term in vivo experiments. Methods: A total of 55 healthy New Zealand rabbits were randomly divided into blank control group, model control group, SP low dose group, SP high dose group, SP blank group, SB low dose group, SB high dose group, SB blank group, dimethyl sulfoxide (DMSO) control group, DMSO blank group, and positive control group. Since the first day of the experiment, each group was administered the corresponding treatment for four weeks continuously. Then, the myocardial c-Jun N-terminal kinase (JNK) and the total protein of p38, protein phosphorylation and its gene expression levels were detected. Results: After intravenous treatment with adriamycin, the myocardial phosphorylate-JNK (p-JNK) and phosphorylate-p38 (p-p38) levels in all groups were increased to varying degrees, of which the model control group increased the most significantly (p < 0.05). Compared with the model control group, the myocardial p-JNK and p-p38 increased more slowly in the SP low dose group, SP high dose group, SB low dose group, SB high dose group and positive control group (p < 0.05), of which the increase in the SP high dose group and the SB high dose group was the slowest (p < 0.05). After four weeks, the total protein and messenger ribonucleic acid of the myocardial JNK and p38 in all groups had no statistically significant difference (p > 0.05). Conclusion: The continuous intravenous injection of SP600125 and SB203580 for four weeks significantly reduced the protein phosphorylation levels of JNK and p38, which provides a practical avenue for the long-term study in vivo.

RESUMEN Objetivo: Explorar los métodos de aplicación de los inhibidores SP600125 y SB203580 de la vía de señalización de la proteína quinasa activada por mitógeno en experimentos in vivo a largo plazo. Métodos: Un total de 55 conejos sanos de Nueva Zelandia fueron divididos aleatoriamente en los grupos siguientes: grupo de control en blanco, grupo de control modelo, grupo de dosis baja SP, grupo de dosis alta SP, grupo en blanco SP, grupo de dosis baja SB, grupo de dosis alta SB, grupo en blanco SB, grupo de control dimetilsulfóxido (DMSO), grupo en blanco DMSO, y grupo de control positivo. Desde el primer día del experimento, a cada grupo se le administró el tratamiento correspondiente por cuatro semanas continuas. Entonces, se detectaron la quinasa c-Jun N-terminal (JNK) miocárdica y la proteína p38 total, así como la fosforilación proteica y sus niveles de expresión génica. Resultados: Después del tratamiento intravenoso con adriamicina, los niveles de fosfo-JNK (p-JNK) y fosfo-p38 (p-p38) del miocardio aumentaron en todos los grupos en diversos grados, siendo el aumento del grupo de control modelo el más significativo (p < 0.05). En comparación con el grupo de control modelo, p-JNK y p-p38 miocárdicos aumentaron más lentamente en el grupo de dosis baja SP, el grupo de dosis alta SP, el grupo de dosis baja SB, el grupo de dosis alta SB, y el grupo de control positivo (p < 0.05). De estos, el aumento en el grupo de dosis alta SP y el grupo de dosis alta SB fue el más lento (p < 0.05). Después de cuatro semanas, la proteína total y el ácido ribonucleico mensajero de JNK y p38 miocárdicos en todos los grupos, no tuvieron diferencias significativas (p > 0.05). Conclusión: La inyección intravenosa continua de SP600125 y SB203580 durante cuatro semanas redujo significativamente los niveles de fosforilación proteica de JNK y p38, lo que proporciona una vía práctica para el estudio a largo plazo in vivo.

A novel homozygous mutation in the solute carrier family 12 member 3 gene in a Chinese family with Gitelman syndrome

Loss of function of mutated solute carrier family 12 member 3 (SLC12A3) gene is the most frequent etiology for Gitelman syndrome (GS), which is mainly manifested by hypokalemia, hypomagnesemia and hypocalciuria. We report the genetic characteristics of one suspicious Chinese GS pedigree by gene sequencing. Complete sequencing analysis of the SLC12A3 gene revealed that both the proband and his elder sister had a novel homozygous SLC12A3 mutation: c.2099T>C and p.Leu700Pro. Moreover, the SLC12A3 genes of his mother and daughter encoded the same mutated heterozygote. It was noted that in this pedigree, only the proband complained about recurrent episodes of bilateral lower limb weakness over 8 years, while his elder sister, mother and daughter did not present symptoms. The inconsistent clinical features of this pedigree implied that besides diverse phenotypes possibly originated from the same genotype, gender difference may also dominate the variant GS phenotypes. Further genetic and proteomic research are needed to investigate the precise mechanisms of GS, including the study of specific ethnicities.