Cardamom extract alleviates tamoxifen-induced liver damage by suppressing inflammation and pyroptosis pathway

Abstract

Tamoxifen (TAM) is extensively used to manage estrogen receptor-positive breast cancer. Despite its effectiveness, its administration can negatively impact various organs, including the liver. This research focused on the effects of TAM on the pyroptotic pathway in the liver and evaluated the potential of cardamom extract (CRDE) to lessen hepatic damage of TAM in female rats. Rats received 45 mg/kg of TAM injections for 10 days, while the groups treated with CRDE received 12 ml/kg of CRDE for 20 days, commencing 10 days before TAM administration. TAM exposure resulted in apparent degenerations in hepatic tissue with inflammatory cell infiltration and loss of architectures. Serum levels of liver enzymes including alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were elevated, along with hepatic oxidative stress, as shown by increased lipid peroxidation with lower levels of reduced glutathione. TAM caused inflammation in the liver tissue as indicated by higher levels of tumor necrosis factor-α and interleukin-6 as well as increased expression of CD68; a phagocytic Kupffer's cells marker. Additionally, the protein expression analysis revealed a high expression of pyroptotic markers including NLRP3-inflammasome, caspase-1, and gasdermin D. Conversely, CRDE treatment effectively neutralized the biochemical, histological, and protein expression alterations induced by TAM. In conclusion, CRDE demonstrated the potential to protect the liver from TAM-induced damage by regulating mechanisms involving oxidative damage, inflammation, and pyroptosis.

Keywords: CD68; Cardamom extract; Liver damage; Liver inflammation; Pyroptosis; Tamoxifen.

Fig. 1

Representative histological staining of the liver. (A) Normal liver with normal architecture and hepatocytes (arrows). (B) Liver from rat received 12 ml/kg CRDE only showing normal hepatocytes and hepatic architecture (arrow). (C) Liver from rat received TAM showing focal patches of degenerations and loss of architectures (stars) that were surrounded by degenerated hepatocytes (arrows). (D) Liver from rat received TAM and 12 ml/kg CRDE showing apparent improvement of hepatic structures, (n = 6), scale bar 100 μm, and amplification ×200.

Fig. 2

Effect of 12 ml/kg CRDE on hepatic levels of oxidative stress markers in TAM-intoxicated female rats. (A) Malondialdehyde (MDA), ****P < 0.0001 normal control compared to TAM group, *P < 0.05 TAM group compared to TAM + CRDE (12 ml/kg). (B) Reduced glutathione (GSH), **P < 0.01 normal control compared to TAM group, *P < 0.05 TAM group compared to TAM + CRDE (12 ml/kg). (C) Superoxide dismutase (SOD) activity, ****P < 0.0001 normal control compared to TAM group, ****P < 0.0001 TAM group compared to TAM + CRDE (12 ml/kg). Values are expressed as mean ± SEM, (n = 6).

Fig. 3

Effect of CRDE on hepatic levels of inflammatory markers in TAM-intoxicated female rats. (A) Tumor necrosis factor-α (TNF-α), ****P < 0.0001 normal control compared to TAM group, ***P < 0.001 TAM group compared to TAM + CRDE (12 ml/kg). (B) Interleukin-6 (IL-6), ****P < 0.0001 normal control compared to TAM group, ****P < 0.0001 TAM group compared to TAM + CRDE (12 ml/kg). Values are expressed as mean ± SEM, (n = 6).

Fig. 4

Effects of CRDE on hepatic CD68 expression in TAM-intoxicated rats. (A) Normal liver with normal number and size of liver phagocytic cells, Kupffer cells (arrows). (B) Liver from rat that received 12 ml/kg of CRDE only showing normal number and size of Kupffer cells as normal control sections (arrows). (C) Liver from rat that received TAM showing marked increases in size and number of Kupffer cells (arrows). (D) Liver from rat received TAM and 12 ml/kg of CRDE showing a marked decrease in size and number of phagocytic cells (arrow), (n = 6), scale bar 50 μm, and amplification ×400.

Fig. 5

Effects of CRDE on hepatic NLRP3 expression in TAM-intoxicated rats. (A) Normal liver with normally low expression of NLRP3. (B) Liver from rat received 12 ml/kg of CRDE only showing no change in the protein expression relative to the control. (C) Liver from rat received TAM showing positive immunoreactive signals of NLRP3 (arrows). (D) Liver from rat received TAM and 12 ml/kg of CRDE showing a decrease in the immunoreactivity (arrow), (n = 6), scale bar 50 μm, and amplification ×400.

Fig. 6

Effects of CRDE on caspase-1 liver expression in TAM-intoxicated rats. (A) Liver sections from normal control rats showed normally low expression of caspase-1. (B) Liver from rat received 12 ml/kg of CRDE only showing no change in caspase-1 protein expression relative to the controls. (C) Liver from rat that received TAM showing a moderate increase in immunoreactive signals of caspase-1 (arrows). (D) Liver from rat received TAM and 12 ml/kg of CRDE showing a decrease in the immunoreactivity (arrow), (n = 6), scale bar 50 μm, and amplification ×400.

Fig. 7

Effects of CRDE on hepatic GSDMD expression in TAM-intoxicated rats. (A) Normal liver with normally mild GSDMD immunoreactivity of hepatocytes (arrows). (B) Liver from rat received 12 ml/kg of CRDE only showing no change in GSDMD immunoreactivity of hepatocytes relative to controls (arrows). (C) Liver from rat received TAM showing strong upregulation of GSDMD immunoreactivity of nuclei and cytoplasm of the hepatocytes (arrow). (D) Liver from rat received TAM and 12 ml/kg of CRDE showing a marked decrease of GSDMD immunoreactivity of hepatocytes (arrow), (n = 6), scale bar 50 μm, and amplification ×400.

Fig. 8

Schematic presentation of CRDE protective effects against TAM-induced oxidative, inflammatory and pyroptosis pathways in the liver. CRDE cardamom extract (CRDE), TAM tamoxifen, NLRP3 inflammasomes, IL-1β interleukin-1β GSDMD, Gasdermin D, and N-GSDMD; N-terminal fragment of GSDMD. Created by BioRender.