Abstract：Puerarin has been proved to have antipyretic effects in animals. However, how puerarin works on the cellular or molecular levels is still poorly understood. The aim of the present study is to determine if puerarin has protective effect on the pig (Sus scrofa) kidney proximal tubular cells (LLC-PK1) subjected to heat stress and its possible underlying mechanisms in vitro. The LLC-PK1 cells were divided into control (C) group, heat stress (HS, 42 ℃) group, puerarin (Pue, 10 µmol/L) group, and heat stress + puerarin (HS+Pue) group. Cell viability was measured by CCK-8; cells apoptosis rate was detected by flow cytometry; mRNA and protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cytochrome c (Cyto c) and apoptosis inducing factor (AIF) were detected by qRT-PCR and Western blot, respectively. Cysteinyl aspartate specific proteinase-9 (Caspase-9) and Caspase-3 activities and total protein content of heat shock protein 72 (HSP72) and HSP90 were detected with the corresponding kits. The results showed that puerarin suppressed Cyto c and AIF mRNA expressions and their proteins released from mitochondria, and decreased both Caspase-9 and Caspase-3 activities of the LLC-PK1 cells under HS. Moreover, puerarin further increased Bcl-2/Bax ratio and HSP72 expressions, and decreased HSP90 expressions of the cells under HS. The results indicated that puerarin attenuates HS-induced apoptosis of the LLC-PK1 cells through inhibiting mitochondrial apoptotic pathway activation, and up-regulating HSP72 expression. This study reveals a part of the mechanism by which puerarin inhibits heat stress-induced damage in LLC-PK1 cells, providing basic materials for the use of puerarin as an effective drug against heat stress in practice.
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