Butyrolactone-I from Marine Fungal Metabolites Mitigates Heat-Stress-Induced Apoptosis in IPEC-J2 Cells and Mice Through the ROS/PERK/CHOP Signaling Pathway

Abstract

Heat stress poses a significant challenge to animal husbandry, contributing to oxidative stress, intestinal mucosal injury, and apoptosis, which severely impact animal health, growth, and production efficiency. The development of safe, sustainable, and naturally derived solutions to mitigate these effects is critical for advancing sustainable agricultural practices. Butyrolactone-I (BTL-I), a bioactive compound derived from deep-sea fungi (Aspergillus), shows promise as a functional feed additive to combat heat stress in animals. This study explored the protective effects of BTL-I against heat-stress-induced oxidative stress and apoptosis in IPEC-J2 cells and mice. Our findings demonstrated that BTL-I effectively inhibited the heat-stress-induced upregulation of HSP70 and HSP90, alleviating intestinal heat stress. Both in vitro and in vivo experiments revealed that heat stress increased intestinal cell apoptosis, with a significant upregulation of Bax/Bcl-2 expression, while BTL-I pretreatment significantly reduced apoptosis-related protein levels, showcasing its protective effects. Furthermore, BTL-I suppressed oxidative stress markers (ROS and MDA) while enhancing antioxidant activity (SOD levels). BTL-I also reduced the expression of p-PERK, p-eIF2α, ATF4, and CHOP, mitigating oxidative and endoplasmic reticulum stress in intestinal cells. In conclusion, BTL-I demonstrates the potential to improve animal resilience to heat stress, supporting sustainable livestock production systems. Its application as a natural, eco-friendly feed additive will contribute to the development of sustainable agricultural practices.

Keywords: Butyrolactone-I (BTL-I); HSP70 and HSP90 expression; endoplasmic reticulum stress; heat stress; intestinal apoptosis; oxidative stress.

Figure 1

Effect of BTL-I on the expression of related factors in heat-shocked IPEC-J2 cells. IPEC-J2 cells were treated with different concentrations of BTL-I (10, 20, or 50 μM) for 24 h and then coincubated with the heat shock (HS) group in a 5% CO₂ incubator at 42 °C for 1.5 h. Cell viability was determined via a CCK8 assay. (A) Effects of BTL-I on the viability of IPEC-J2 cells; (B) effects of heat shock treatment on the viability of IPEC-J2 cells; (C) effects of BTL-I and heat shock cotreatment on IPEC-J2 cells; and (D–F) the expression levels of the heat shock proteins HSP70 and HSP90 were detected via Western blotting. The results are expressed as the means ± SEMs. ^# p < 0.05, ^## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the HS group.

Figure 2

Effects of BTL-I on IPEC-J2 cell apoptosis after heat shock. (A) Flow cytometry analysis of the cell apoptosis rate; (B,C) qPCR analysis of Bax and Bcl-2; (D,E) Western blotting analysis of Bax and Bcl-2; and (F,G) IF analysis of Bax and Bcl-2; the red fluorescence represents Bcl-2 and Bax protein expression, and the blue fluorescence corresponds to the cell nuclei; the results are expressed as the means ± SEMs. ^# p < 0.05, ^## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the HS group.

Figure 3

Effect of BTL-I on the ROS/PERK/CHOP signaling pathway in IPEC-J2 cells subjected to heat shock. (A–C) ELISA analysis of the levels of the oxidative markers ROS, MDA, and SOD; (D,E) qPCR analysis of the levels of the PERK/CHOP signaling pathway markers ATF4 and CHOP; (F–J) Western blotting analysis of the levels of the PERK/CHOP signaling pathway markers p-PERK, PERK, p-eIF2α, eIF2α, ATF4, and CHOP; and (K) IF analysis of the levels of the PERK/CHOP signaling pathway marker ATF4. The results are expressed as the means ± SEMs. ^# p < 0.05, ^## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the HS group.

Figure 4

Effects of ROS scavengers on the PERK/CHOP signaling pathway and apoptosis in heat-shocked IPEC-J2 cells. IPEC-J2 cells were treated with BTL-I (50 μM) or NAC (1 mM) for 24 h or 3 h, respectively, and then placed together with the heat shock (HS) group in a cell incubator at 42 °C and 5% CO₂ for 1.5 h. (A) ELISA analysis of the oxidative marker ROS. (B–G) Western blot analysis of the expression of the PERK/CHOP signaling pathway markers p-PERK, PERK, p-eIF2α, eIF2α, ATF4, and CHOP. (H–L) Western blot analysis of the expression of the apoptosis markers Bax/Bcl-2 and procaspase 3. The results are expressed as the means ± SEMs. ^# p < 0.05, ^## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the HS group.

Figure 5

Effect of ROS scavengers on apoptosis in heat-shocked IPEC-J2 cells. (A,B) IF analysis of the apoptosis markers Bax and Bcl-2.

Figure 6

Effect of an ER stress inhibitor on the apoptosis of heat-shocked IPEC-J2 cells. IPEC-J2 cells were treated with BTL-I (50 μM) or 4-PBA (1 mM) for 24 h or 3 h, respectively, and then placed together with the heat shock (HS) group in a cell incubator at 42 °C and 5% CO₂ for 1.5 h. (A–F) Western blot analysis of the expression of the PERK/CHOP signaling pathway markers p-PERK, PERK, p-eIF2α, eIF2α, ATF4, and CHOP. (G–K) Western blot analysis of the expression of the apoptosis markers Bax/Bcl-2 and procaspase 3. The results are expressed as the means ± SEMs. ^# p < 0.05, ^## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the HS group.

Figure 7

Effect of an endoplasmic reticulum inhibitor on the apoptosis of heat-shocked IPEC-J2 cells. (A,B) IF analysis of the apoptosis markers Bax and Bcl-2.

Figure 8

Protective effect of BTL-I on heat-stressed mice. The normal control group (CON) was exposed to 24 ± 1 °C and treated with PBS (0.2 mL), the heat stress group (HS) was subjected to 40 ± 1 °C for 4 h per day, the HS group had a low oral BTL-I concentration (1 mg/kg) (LOW), and the HS group had a high oral BTL-I concentration (5 mg/kg) (HIGH). Clinical changes were recorded, and colon tissues were collected. (A–D) qPCR and Western blotting analysis of the effects of BTL-I on HSP70 and HSP90 proteins; (E) water intake of the mice during the test; (F) body weight changes in the mice during the test; (G) body temperature changes; and (H,I) colon length of the mice during the test; the results are expressed as the means ± SEMs. ^# p < 0.05, ^## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the HS group.

Figure 9

Effects of BTL-I on apoptosis in heat-stressed mice. (A) The effects of BTL-I on the expression of the apoptotic marker Bax were determined by qPCR. (B–C) The effects of BTL-I on the expression of the apoptosis markers Bax, Bcl-2, and Pro-Caspase 3 were determined by Western blotting. (D) Immunofluorescence staining for apoptosis was performed via the TUNEL assay. The results are expressed as the means ± SEMs. ^## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the HS group.

Figure 10

Effects of BTL-I on the ROS/PERK/CHOP signaling pathway in heat-stressed mice. (A–C) The effects of BTL-I on the expression of oxidative cytokines were determined by ELISA; (D,E) qPCR analysis of the expression of the PERK/CHOP signaling pathway markers ATF4 and CHOP; and (F–J) Western blotting analysis of the expression of the PERK/CHOP signaling pathway markers p-PERK, PERK, p-eIF2α, eIF2α, ATF4, and CHOP; the results are expressed as the means ± SEMs. ^# p < 0.05, ^## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the HS group.

Figure 11

Mouse experiment.