Ketamine induces endoplasmic reticulum stress in rats and SV-HUC-1 human uroepithelial cells by activating NLRP3/TXNIP aix

Abstract

Many clinical studies have been conducted on ketamine-associated cystitis. However, the underlying mechanisms of ketamine-associated cystitis still remain unclear. Bladder tissues of rats were stained by Hematoxylin and Eosin (HE). The viability of human uroepithelial cells (SV-HUC-1 cells) was determined by cell counting kit-8 (CCK-8). Apoptosis and reactive oxygen species (ROS) were examined by flow cytometry. Additionally, the expressions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β and IL-18 were respectively determined by reverse transcription quantitative (RTq)-PCR and enzyme-linked immunosorbent assay (ELISA). The mRNA and protein levels of B-cell lymphoma/leukemia-2 (Bcl2), Bcl-2-associated X protein (Bax), cleaved caspase 3, glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP), NOD-like receptor 3 (NLRP3), thioredoxin-interacting protein (TXNIP), Catalase and MnSOD were examined by RT-qPCR and Western blot. Small interfering RNA target TXNIP transfection was performed using Lipofectamine™ 2000. We found that ketamine effectively damaged bladder tissues of rats and promoted apoptosis through regulating the expression levels of GRP78, CHOP, Bcl-2, Bax and cleaved Caspase-3 proteins in vivo and in vitro. NLRP3 inflammatory body and TXNIP were activated by ketamine, which was supported by the changes in TNF-α, IL-6, IL-1 and IL-18 in vivo and in vitro. Furthermore, knocking down TXNIP reversed the effects of ketamine on apoptosis and NLRP3 inflammatory body in SV-HUC-1 cells. Meanwhile, the changes of Catalase and MnSOD showed that ROS was enhanced by ketamine, however, such an effect was ameliorated by down-regulation of TXNIP in SV-HUC-1 cells. Ketamine promoted cell apoptosis and induced inflammation in vivo and in vitro by regulating NLRP3/TXNIP aix.

Keywords: Ketamine; NOD-like receptor 3/Thioredoxin-interacting protein; SV-HUC-1 cells; apoptosis.

Figure 1. ERS-induced apoptosis and NLRP3/TXNIP were activated by ketamine

(A) The bladder tissues of rats treated with lower and higher doses of ketamine were stained by HE staining. (B,C) The protein levels of GRP78, CHOP, Bax, Bcl-2, cleaved caspase-3, NLRP3 and TXNIP were determined (B) and quantified (C) by Western blot. (D) The mRNA levels of GRP78, CHOP, Bcl-2, Bax, NLRP3 and TXNIP were determined by RT-qPCR. *P<0.05, **P<0.01 vs. control.

Figure 2. Cell apoptosis and NLRP3/TXNIP were induced by ketamine in SV-HUC-1 cells

(A) The viability of SV-HUC-1 cells treated with 0.01, 0.1 and 1 mmol/l ketamine for 24, 48 and 72 h were determined by CCK-8. (B) Ketamines at 0.01, 0.1 and 1 mmol/l was used to treat SV-HUC-1 cells apoptosis, which was analyzed by flow cytometry. (C) TNF-α and IL-6 mRNA levels were analyzed by RT-qPCR. (D,E) The protein levels of GRP78, CHOP, NLRP3 and TXNIP were determined (D) and quantified (E) by Western blot. (F) The mRNA levels of GRP78, CHOP, NLRP3 and TXNIP were determined by RT-qPCR. (G) The protein levels of IL-1β and IL-18 were measured by ELISA. *P<0.05, **P<0.01 vs. control.

Figure 3. Knockdown of TXNIP inhibited SV-HUC-1 cells apoptosis and inflammation

(A,B) SV-HUC-1 cells apoptosis was analyzed (A) and quantified (B) by flow cytometry. (C) TNF-α and IL-6 mRNA levels were analyzed by RT-qPCR. (D,E) The protein levels of GRP78, CHOP, Bax, Bcl2, cleaved caspase-3, NLRP3 and TXNIP were determined (D) and quantified (E) by Western blot. (F) The mRNA levels of GRP78, CHOP, Bax, Bcl2, NLRP3 and TXNIP were determined by RT-qPCR. (G) The protein levels of IL-1β and IL-18 were measured by ELISA. *P<0.05, **P<0.01 vs. control, ^#P<0.05, ^##P<0.01 vs. KET, ^&P<0.05, ^&&P<0.01 vs. NC+KET.

Figure 4. Knockdown of TXNIP reversed ROS production caused by ketamine in SV-HUC-1 cells

(A) SV-HUC-1 cells ROS production was analyzed by flow cytometry. (B,C) The protein levels of Catalase and MnSOD were determined (B) and quantified (C) by Western blot. (D) The mRNA levels of Catalase and MnSOD were assessed by RT-qPCR. **P<0.01 vs. control, ^##P<0.01 vs. KET, ^&&P<0.01 vs. NC+KET.