Abstract:

Objective To explore the role of secreted phosphoprotein 1 （SPP1） and myeloid differentiation primary response 88 （MYD88） in morphine-induced cardiomyocyte apoptosis. Methods A morphine addiction model was established in Sprague-Dawley （SD） rats. Twelve SD rats were randomly assigned to the normal saline （NS） group or the morphine-dependent （DAM） group. Histopathological analysis was employed to observe and compare myocardial tissue morphology between the two groups. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） staining was performed to assess the number of apoptotic cells in each group. The expression levels of SPP1 and MYD88 were evaluated using immunohistochemistry. Quantitative real-time poly merase chain reaction （RT-qPCR） and Western blot were used to detect the mRNA and protein expression of SPP1， MYD88， Bax， Bcl2， Caspase-3， and Caspase-9. Simultaneously， Western blot analysis was used to detected the expression of Cleaved Caspase-3 and Cleaved Caspase-9 proteins. In vitro， SPP1 expression was knocked down in primary neonatal rat cardiomyocytes （NRCMs）， and cells were divided into three groups： control （CON）， morphine treated （DA）， and shSPP1#3 + DA. Cell viability was assessed using the CCK-8 assay， and apoptosis rates were determined by flow cytometry. Results HE and TUNEL staining of myocardial tissues from morphine-addicted SD rats revealed that， compared with the NS group， myofibrils in the DAM group exhibited partial disruption and a significant increase in apoptotic cells （P < 0.05）. Western blot and RT-qPCR analyses demonstrated that， relative to the NS group， the mRNA and protein levels of SPP1， MYD88， Bax， Caspase-3， and Caspase-9 were significantly upregulated in the DAM group （P < 0.05）， whereas Bcl2 expression was significantly downregulated at both mRNA and protein levels （P < 0.05）， and the protein expression levels of Cleaved Caspase-3 and Cleaved Caspase-9 were also increased. with all differences being statistically significant. In NRCMs following morphine intervention， cell viability in the DA group was markedly reduced compared to the CON group （P < 0.05）， accompanied by a significant increase in apoptosis rate （P < 0.05）. Consistently， Western blot and RT-qPCR results showed elevated mRNA and protein expression of SPP1， MYD88， Bax， Caspase-3， and Caspase-9 in the DA group （P < 0.05）， along with decreased Bcl2 expression （P < 0.05）. The protein expression levels of Cleaved Caspase-3 and Cleaved Caspase-9 were elevated simultaneously. In contrast， the shSPP1#3 + DA group exhibited opposing trends compared to the DA group， with statistically sig nificant differences （P < 0.05）. Conclusion SPP1 and MYD88 play critical roles in mediating morphine-induced cardiomyocyte apoptosis， and silencing SPP1 has been shown to significantly reduce the extent of cardiomyocyte apoptosis following morphine exposure.

Key words: cardiomyocytes, apoptosis, diacetylmorphine, SPP1, MYD88