ROCK2敲低通过NF-κB/NLRP3通路减轻LPS诱导的肾小管上皮细胞炎症损伤和凋亡

Abstract

Rho‑associated protein kinase 2 (ROCK2) is an important regulator of the inflammatory response and has been reported to serve a role in sepsis. The present study aimed to investigate whether ROCK2 served a role in sepsis‑associated acute kidney injury (S‑AKI). HK‑2 cells were stimulated with lipopolysaccharide (LPS) to simulate S‑AKI in vitro. Subsequently, the change in ROCK2 expression levels were determined. ROCK2 in LPS‑induced HK‑2 cells was knocked down using short hairpin RNA‑ROCK2, in the absence or presence of phorbol 12‑myristate 13‑acetate (PMA), an activator of NF‑κB. Cell viability, cytotoxicity, inflammation and apoptosis were assessed using MTT, lactate dehydrogenase (LDH) release, reverse transcription‑quantitative PCR, ELISA, TUNEL and western blotting assays. The protein expression levels of proteins involved in the NF‑κB/NLR family pyrin domain containing 3 (NLRP3) signaling pathway were also assessed using western blotting. The results demonstrated that ROCK2 was upregulated in HK‑2 cells upon LPS treatment. LPS also reduced cell viability, promoted LDH activity and increased TNF‑α, IL‑6 and IL‑1β mRNA expression levels and concentrations. Apoptosis was also induced by LPS as indicated by an increase in the proportion of TUNEL‑positive cells, decreased Bcl‑2 protein expression levels and increased cleaved caspase‑3 and cleaved poly (ADP‑ribose) polymerase protein expression levels. However, ROCK2 knockdown in LPS‑induced HK‑2 cells reversed cell viability damage and inhibited LDH activity, the generation of pro‑inflammatory cytokines and apoptosis caused by LPS. Furthermore, ROCK2 knockdown inhibited the LPS‑induced expression of phosphorylated‑NF‑κB p65, NLRP3, apoptosis‑associated speck‑like protein containing a CARD and caspase‑1 p20. PMA treatment reversed all the aforementioned effects of ROCK2 knockdown on LPS‑treated HK‑2 cells. Therefore, ROCK2 knockdown may alleviate LPS‑induced HK‑2 cell injury via the inactivation of the NF‑κB/NLRP3 s