Yonsei Med J.  2019 Dec;60(12):1195-1202. 10.3349/ymj.2019.60.12.1195.

Propofol Attenuates Hypoxia/Reoxygenation-Induced Apoptosis and Autophagy in HK-2 Cells by Inhibiting JNK Activation

• Wang H
• Peng X
• Huang Y
• Xiao Y
• Wang Z
• Zhan L

• KMID: 2463799
• DOI: http://doi.org/10.3349/ymj.2019.60.12.1195

Abstract

PURPOSE
The aim of this study was to investigate whether propofol could attenuate hypoxia/reoxygenation-induced apoptosis and autophagy in human renal proximal tubular cells (HK-2) by inhibiting JNK activation.
MATERIALS AND METHODS
HK-2 cells were treated with or without propofol or JNK inhibitor SP600125 for 1 hour and then subjected to 15 hours of hypoxia and 2 hours of reoxygenation (H/R). Cell viability and LDH release were measured with commercial kits. Cell apoptosis was evaluated by flow cytometry. The expressions of p-JNK, cleaved-caspase-3, Bcl-2, and autophagy markers LC3 and p62 were measured by Western blot or immunofluorescence.
RESULTS
HK-2 cells exposed to H/R insult showed higher cell injury (detected by increased LDH release and decreased cell viability), increased cell apoptosis index and expression of cleaved-caspase-3, a decrease in the expression of Bcl-2 accompanied by increased expression of p-JNK and LC3II, and a decrease in expression of p62. All of these alterations were attenuated by propofol treatment. Similar effects were provoked upon treatment with the JNK inhibitor SP600125. Moreover, the protective effects were more obvious with the combination of propofol and SP600125.
CONCLUSION
These results suggest that propofol could attenuate hypoxia/reoxygenation induced apoptosis and autophagy in HK-2 cells, probably through inhibiting JNK activation.

Keyword

Propofol; HK-2 cells; apoptosis; autophagy

MeSH Terms

Anoxia
Apoptosis*
Autophagy*
Blotting, Western
Cell Survival
Flow Cytometry
Fluorescent Antibody Technique
Humans
Propofol*
Propofol

Figure

• 

  Fig. 1 Effects of propofol on hypoxia and reoxygenation (H/R)-induced cytotoxicity to HK-2 cells. Cell viability of the control group with neither H/R injury nor propofol pretreatment was used as a 100% benchmark. H/R injury led to reduced cell viability (##p<0.01 against the control group), and propofol pretreatment alleviated decreases in cell viability induced by H/R injury (*p<0.05 and **p<0.01 against the H/R injury group). No significant difference was observed between pretreatments with 50 µM and 100 µM propofol (Pro 10, 10 µM; Pro 25, 25 µM; Pro 50, 50 µM; Pro 100, 100 µM).

• 

  Fig. 2 Effects of propofol on LDH leakage in HK-2 cells. LDH levels were measured for the same control and experimental groups as in Fig. 1. Consistently, hypoxia and reoxygenation (H/R) injury led to significantly higher levels of LDH (##p<0.01 against the control group), and propofol pretreatment alleviated increases in LDH caused by H/R injury (**p<0.01 against H/R group).

• 

  Fig. 3 Effects of propofol on apoptosis of HK-2 cells. Flow cytometry was conducted on the same control and experimental groups as in Fig. 1 (A: Control group; B: Hypoxia and reoxygenation (H/R) group; C: Pro 10 group; D: Pro 25 group; E: Pro 50 group; F: Pro 100 group). (G) Flow cytometric analysis for (A–F) was carried out as described in the Materials and Methods section. H/R injury increased the number of apoptotic cells (##p<0.01 against the control group), and propofol pretreatment alleviated the observed increases (*p<0.05 and **p<0.01 against H/R group).

• 

  Fig. 4 Potential synergistic effect of propofol and SP600125 on alleviating cell apoptosis induced by hypoxia and reoxygenation (H/R) injury. (A–E) Representative figures of flow cytometry results for (A) the control group with neither H/R injury nor propofol/SP600125 pretreatment, (B) the H/R injury group with no pretreatment, (C) the SP600125 group pretreated with 10 µM SP600125 prior to H/R injury, (D) the Pro 50 group pretreated with 50 µM propofol prior to H/R injury, and (E) the Pro 50+SP600125 group pretreated with a combination of 50 µM propofol and 10 µM SP600125 prior to H/R injury. (F) Flow cytometric analysis for (A–E) was carried out as described in the Materials and Methods section. H/R injury increased cell apoptosis significantly (##p<0.01 against the control group), while a combination of propofol and SP600125 attenuated the increase in a potentially synergistic manner (*p<0.05 against H/R group; **p<0.01 against H/R group; ▲p<0.05 against Pro 50 group).

• 

  Fig. 5 Effects of propofol and SP600125 on the protein expression levels of JNK, p-JNK, caspase-3, cleaved-caspase-3, and Bcl-2 in HK-2 cells. Western blots were performed for the same control and experimental groups as in Fig. 4 to measure expression levels of the above-mentioned proteins. (A) Raw data. (B–D) Hypoxia and reoxygenation (H/R) injury increased the protein expression levels of p-JNK and cleaved-caspase-3, and decreased those of Bcl-2. Both SP600125 and propofol attenuated the changes in expression levels, and a combination of both alleviated the changes further (##p<0.01 against the control group; *p<0.05 and **p<0.01 against the H/R group; ▲p<0.05 and ▲▲p<0.01 against the Pro 50 group).

• 

  Fig. 6 Effects of propofol and SP600125 on autophagy induced by hypoxia and reoxygenation (H/R) injury. The same control and experimental groups as in Fig. 4 were subjected to fluorescence microscopy (A–C) and Western blots (D–F) to measure the protein expression levels of LC3II and p62 (autophagy markers). Both techniques showed that H/R injury increased the protein expression levels of LC3II and decreased those of p62. Both SP600125 and propofol attenuated the changes, and their alleviating effects were stronger when applied together (##p<0.01 against the control group; *p<0.05 and **p<0.01 against the H/R group; ▲p<0.05 against the Pro 50 group).

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