Volume 29, Issue 5, 2020
DOI: 10.24205/03276716.2020.1128
Mechanism of Wenyang Zhenshuai Granules in protecting injured H9C2 cardiomyocytes based on the exosomes-mediated miR-155/p38MAPK signal pathway
Abstract
Purpose: To discuss about the mechanism of Wenyang Zhenshuai Granules in protecting injured H9C2 cardiomyocytes based on the exosomes-mediated miR-155/p38MAPK signal pathway.
Methods: The experiments are divided into two steps. Adriamycin (ADR) induced cardiomyocyte injury model was used in experiment 1. Normal group (N), ADR group (A), ADR + control serum group (A + B), ADR + Wenyang Zhenshuai-containing serum group (A + W), ADR + enalapril-containing serum group (A + Y) were set up. Cell status was monitored by microscope and MTT method. After 45 hours, p38MAPK and its phosphorylation expression were detected by WB method, p38MAPK mRNA and miR-155 content were detected by PCR method. In experiment 1, exosomes in A + W and A + Y groups were isolated after culture. After identification, fluorescence labeling was completed by transmission electron microscope and WB method. Co-culture system was established with cardiomyocytes injury model. Normal group (N), ADR group (A), ADR + A + W exosomes group (A + A + W) and ADR + A + Y exosomes group (A + A + Y) were set up. The process of exosomes aggregating and entering cardiomyocytes was observed by laser confocal system. After 45 hours, the cell viability, protein and gene were detected by light microscope, MTT, WB and PCR methods. The detection indexes were the same as those in experiment 1.
Results: Experiment 1: Light microscope showed vacuolar degeneration of modelling group cells, and degeneration of A + W group and A + Y group was improved. MTT experiment showed that compared with N group, OD value in modelling group decreased significantly. Compared with A group, OD values in A + W group and A + Y group increased significantly. The difference was statistically significant (p < 0.05 or p < 0.01). The results of WB and PCR showed that the mRNA of p38MAPK, p-p38MAPK and p38MAPK was up-regulated and the expression of miR-155 was down-regulated in the modelling group compared with that in the N group; compared with A group, the mRNA of p38MAPK, p-p38MAPK and p38MAPK were down-regulated and the expression of miR-155 was up-regulated in A + W group and A + Y group. The difference was statistically significant (p < 0.05 or p < 0.01). Experiment 2: Laser confocal system observed that exosomes can aggregate and enter damaged cardiomyocytes. MTT experiment showed that compared with N group, OD value in modelling group decreased significantly. Compared with A group, OD values in A + A + W group and A + A + Y group increased significantly. The difference was statistically significant (p < 0.05 or p < 0.01). The results of WB and PCR showed that the mRNA of p-p38MAPK and p38MAPK was up-regulated and the expression of miR-155 was down-regulated in the modelling group compared with that in the N group. Compared with A group, A + W group and A + Y group could down-regulate the mRNA of p-p38MAPK and p38MAPK and up-regulate miR-155 expression. The difference was statistically significant (p < 0.05 or p < 0.01).
Conclusion: Wenyang Zhenshuai Granules can protect damaged cardiomyocytes by up-regulating miR-155 and down-regulating target cell p-p38MAPK mediated by exosomes.
Keywords
Exosomes; Wenyang Zhenshuai Granules; H9C2 cardiomyocytes