Molecular mechanism of empagliflozin cardioprotection in 5-fluorouracil (5-FU)-induced cardiotoxicity via modulation of SGLT2 and TNFα/TLR/NF-κB signaling pathway in rats

Abstract

One of the commoly used chemotherapeutic agents is 5-Fluorouracil (5-FU). Unfortunately, the clinical administration of 5-FU is complicated with serious cardiotoxic effects and the safe use becomes an urgent task in cardio-oncology. Till now, there are no studies discussed the role of empagliflozin (EMP) against 5-FU cardiotoxicity. Thus, we investigated this effect and the involved mechanisms in 5-FU induced heart injury. Forty male rats of Wistar albino species were used and divided randomly into four groups. Group I is the control group, group II is EMP given group, group III is 5-FU cardiotoxic group and group IV is 5-FU plus EMP group. 5-FU (150 mg/kg) was administered as a single intraperitoneal (i.p.) dose on 1st day to induce cardiotoxicity with or without EMP (30 mg/kg/d) orally for 5 days. The dose of 5-FU is relevant to the human toxic dose. Our data showed that 5-FU given group caused cardiotoxicity with significant increase of serum cardiac enzymes, toll like receptors, enhancement of nuclear factor kappa B (NF-κB), interleukin1β (IL1β), IL6, myeloid-differentiation-factor 88 (MYD88), heart weight, malondialdehyde (MDA), tumor-necrosis-factor-alpha (TNFα), sodium glucose co-transporter 2 (SGLT2), P53 and caspase3 expression with clear histopathological features of cardiotoxicity. Moreover, there is a significant decrease in reduced glutathione (GSH) and total antioxidant capacity (TAC). Interestingly, co-administration of EMP could ameliorate 5-FU induced biochemical and histopathological changes. This effect may be due to modulation of SGLT2, decreasing inflammation, oxidative stress and apoptosis with downregulation of an essential inflammatory cascade that mediates 5-FU cardiotoxicity; TNFα/TLR/NF-κB.

Supplementary information: The online version contains supplementary material available at 10.1007/s43188-023-00204-1.

Keywords: 5-Fluorouracil; Cardiotoxicity; Empagliflozin; Toll like receptor.

Fig. 1

a–d Western blotting evaluation of caspase3, IL1β, IL6, TLR2 expression. Our data found significant increase of caspase3, IL1β, IL6, TLR2 expression in 5-fluorouracil (5-FU) given group compared to control group. However, empagliflozin (EMP) administration plus 5-fluorouracil (5-FU) revealed significant decrease of these parameters in comparison to 5-fluorouracil (5-FU) given rats. Values represents means ± SEM of 10 animals in each group. It is considered significantly different if p value less than 0.05. ^aSignificant difference if compared to control, ^bsignificant difference in comparison to 5-fluorouracil group, ^csignificant difference compared to 5-fluorouracil treated group. CON represents control group, EMP is empagliflozin group, 5-FU represents 5-fluorouracil group, 5-FU + EMP is 5-fluorouracil plus empagliflozin group. IL1β is interleukin 1β, IL6 is interleukin 6, TLR2 is toll like receptor 2

Fig. 2

Histopathological evaluation results. Sections of both control group and empagliflozin (EMP) given group revealed preserved integrity of striated cardiac muscle fibres having acidophilic cytoplasm with central oval nuclei (green arrow) (a & b). The 5-fluorouracil (5-FU) cardiotoxic group showed disruption of architecture with loss of cardiac muscle striations, areas of necrotic cardiac tissue (black arrow), congested dilated blood vessels (red arrow) and inflammatory cellular infiltration (blue arrow) (c). There is significant improvement of the histopathological abnormalities observed in empagliflozin (EMP) co-administered group with restoration of cardiac muscle integrity (d). (X200). (Scale bar = 100 µm)

Fig. 3

Evaluation of MYD88 immunoreactivity. Sections of both control and empagliflozin (EMP) given group showed weak positivity in the cytoplasm of cardiac muscles (a, b). Meanwhile, 5-fluorouracil (5-FU) administered group revealed strong immunoexpression in almost all the stained areas (c). Weak positivity was observed in the empagliflozin (EMP) co-administered group (d). (X200) (Scale bar = 100 µm). Semiquantitative analysis of MYD88 immunoexpression: Data revealed that the immunoexpression significantly increased in the 5-fluorouracil (5-FU) group compared to the control group. However, co-administration of empagliflozin (EMP) could significantly decrease its immunoexpression if compared to fluorouracil (5-FU) group (e). Values represents means ± SEM of 10 animals in each group. It is considered significantly different if p value less than 0.05. ^aSignificant difference if compared to control, ^bsignificant difference in comparison to 5-fluorouracil group, ^csignificant difference compared to 5-fluorouracil treated group. CON represents control group, EMP is empagliflozin group, 5-FU represents 5-fluorouracil group, 5-FU + EMP is 5-fluorouracil plus empagliflozin group. MYD88 is myeloid differentiation factor 88

Fig. 4

Evaluation of TLR5 immunoreactivity. Negative immunostaining was detected in both control and empagliflozin (EMP) given groups (a, b). On the other hand, the 5-fluorouracil (5-FU) given group showed strong positive immunoexpression (c). Meanwhile, the empagliflozin (EMP) co-administrated group exhibited weak immunostaining (d) (X200) (Scale bar = 100 µm). Semiquantitative analysis of TLR5 immunoexpression: Semiquantitative analysis showed that there is a significant increase of TLR5 immunoexpression in 5-fluorouracil (5-FU) group compared to control group. However, there is a significant decrease in its immunoexpression in empagliflozin (EMP) co-administered group compared to 5-fluorouracil (5-FU) group (e). Values represents means ± SEM of 10 animals in each group. It is considered significantly different if p value less than 0.05. ^aSignificant difference if compared to control, ^bsignificant difference in comparison to 5-fluorouracil group, ^csignificant difference compared to 5-fluorouracil treated group. CON represents control group, EMP is empagliflozin group, 5-FU represents 5-fluorouracil group, 5-FU + EMP is 5-fluorouracil plus empagliflozin group. TLR5 is toll like receptor 5