ASK1 Inhibitor NQDI‑1 Decreases Oxidative Stress And Neuroapoptosis Via The ASK1/p38 And JNK Signaling Pathway in Early Brain Injury After Subarachnoid Hemorrhage in Rats Part 2

Discussion

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In the present study, protein expression levels of ASK1 and p‑ASK1 were elevated following SAH. ASK1 inhibitor NQDI‑1 improved short‑ and long‑term neurological function after SAH and decreased oxidative stress and neuronal apoptosis via inhibition of ASK1 phosphorylation and the ASK1/p38 and  JNK signaling pathway in EBI following SAH.

ASK1 is a member of the MAP3K family and its activation in response to numerous types of cellular stress mediates different types of cellular damage (31). For certain diseases involving oxidative stress and apoptosis, decreased protein expression of ASK1 or inhibition of its phosphorylation may have a beneficial role by decreasing oxidative stress and apoptosis (32). To the best of our knowledge, however, the role and underlying mechanism of action of ASK1 and its inhibitor NQDI‑1 have not previously been reported in SAH. The present study demonstrated that the protein expression levels of ASK1 and p‑ASK1 were significantly increased following  SAH modeling, which suggested that ASK1 may serve a role in EBI after SAH. Furthermore, ASK1 was co‑expressed in neurons, microglia, and astrocytes, which suggested a wide role for ASK1 in a variety of neuronal cells following SAH.

NQDI‑1 is a specific inhibitor of ASK1 and its functional role has been previously demonstrated using in vitro kinase assays (11,33). In a mouse model of acute pancreatitis, NQDI‑1   decreases pancreatic follicular cell necrosis through the decrease of ROS production and receptor-interacting serine/threonine kinase 3 and p‑mixed lineage kinase domain‑like pseudo ki‑  nase protein expression levels (34). In ischemic brain injury,  NQDI‑1 inhibition of ASK1 decreases matrix metalloproteinase 9 activity and subsequent neuronal apoptosis in brain endothelial cells (35). In the present study, different concentrations of NQDI‑1 were injected intracerebroventricularly, which significantly improved the modified Garcia and balance beam test score, which suggested that NQDI‑1 led to improvements in short‑term neurological function following SAH.

For long‑term neurological function, two methods were used to assess function over different periods (36). The  Rotarod test was used to assess the coordinated balance of locomotion on days 7, 14, and 21. At initial speeds of 5 or  10 rpm, the fall latency of rats in the SAH + vehicle group was significantly shorter compared with that in the sham group, and treatment with NQDI‑1 led to a significant increase. The  Morris water maze test was used to assess the spatial memory and learning ability of rats during the fourth week. During the training phase of days 1‑5, the SAH group rats swam for significantly longer times and further distances compared with the sham group and NQDI‑1 treatment decreased this phenomenon. During the testing phase, no significant differences were demonstrated in the swimming speed between groups of rats.  After the removal of the platform, the time spent exploring the target quadrant was observed and counted for each group of rats. Following SAH, rats searched for the target quadrant for a shorter time, which indicated that SAH modeling led to more ambiguous spatial localization and memory in rats and impaired long‑term memory capacity, whereas NQDI‑1 treat‑  ment demonstrated improvements in these outcomes. The Rotarod experiment and the Morris water maze test results suggested that NQDI‑1 improved long‑term neurological function after  SAH.

Oxidative stress and neuroapoptosis are key pathological changes of EBI following SAH (37). In steady‑state cells,  ROS are primarily byproducts of respiration produced by the mitochondrial electron respiratory chain and moderate levels of ROS repair damaged DNA and serve a physiological role in the promotion of cell survival (38). Upon SAH, due to the autoxidation of blood in the subarachnoid space, ROS catalysis by heme and intracellular mitochondrial dysfunction, electrons escape into the cytoplasm, and the antioxidant system is insufficient to compensate, which results in the accumulation of large amounts of ROS in neuronal cells (39), which leads to oxidative stress damage. Therefore, therapeutic strategies that target oxidative stress and apoptosis may be considered effective therapeutic directions following SAH. In the present study, ROS and oxidative stress of brain tissues were assessed using DHE, which is oxidized into ethidium and produces a red fluorescent signal (40). The percentage of DHE‑positive cells significantly increased in the SAH + vehicle group, whereas a  decrease was demonstrated in the percentage of DHE‑positive cells in the SAH + NQDI‑1 group, which suggested that  NQDI‑1 decreased oxidative stress. Apoptosis was assessed using TUNEL and the percentage of TUNEL‑positive neuronal cells following SAH modeling significantly increased,   whereas NQDI‑1 significantly decreased the percentage of  TUNEL‑positive neuronal cells. It may be hypothesized that treatment NQDI‑1 decreased oxidative stress and apoptosis in  EBI following SAH.

The effect of NQDI‑1 on ASK1 and the potential underlying molecular mechanism was evaluated. The effects of  NQDI‑1 on protein expression levels of ASK1 and p‑ASK1   were first assessed. The ratio of p‑ASK1/ASK1 did not change following SAH, which suggested that changes in the phosphorylation of ASK1 were similar to those of ASK1 protein expression levels. Treatment with NQDI‑1 demonstrated a   significant decrease in p‑ASK1 protein expression levels but demonstrated no effect on ASK1 compared with the SAH  + vehicle group, which suggested that NQDI‑1 exerted its neuroprotective effects primarily via inhibition of ASK1 phosphorylation. Subsequently, protein expression levels of both   p‑ASK1 and ASK1 were knocked down using ASK1 siRNA, following which protein expression levels of p‑p38 and p‑JNK were significantly decreased, which suggested that ASK1 was activated primarily via phosphorylation.

The p38, MAPK, and JNK signaling pathways are widely expressed in brain tissue (41). Activated p38, MAPK, and  JNK enhance tumor necrosis factor‑induced apoptosis, participate in the Fas/FasL system, phosphorylate P53, and induce mitochondrial translocation of BAX and other pathways to promote apoptosis (42). Inhibition of the phosphorylation activation of p38 MAPK and JNK decreases oxidative stress and neuronal apoptosis, alleviates EBI, and improves the prognosis of the SAH rat model (43). After injection of the p38 inhibitor BMS‑582949, the WB results demonstrated that it significantly inhibited the phosphorylation of p38 and significantly downregulated expression levels of oxidative stress and apoptosis‑associated proteins. However,  BMS‑582949 did not cause a significant difference in the protein expression levels of p‑ASK1, ASK1, or p‑JNK.  Similarly, the JNK inhibitor SP600125 significantly inhibited the phosphorylation of JNK but demonstrated no significant effect on p‑ASK1, ASK1, or p‑p38 protein expression levels.  These results suggested that p38 and JNK were downstream of p‑ASK1 and that ASK1 caused oxidative stress and apoptosis following SAH via phosphorylation‑activated p38   and JNK.

There were certain limitations associated with the present study. Firstly, NQDI‑1 was only administered once via intracerebroventricular injection 1 h after SAH; therefore,  the current study was not suitable to determine the optimal therapeutic window for NQDI‑1 treatment and future studies are required to address this issue. Secondly, the present study was a pilot study to evaluate the effect of inhibition of ASK1 on neurological function and to explore whether NQDI‑1 had a therapeutic effect following SAH. The role of ASK1 in astrocytes or microglia and the pharmacokinetics of NQDI‑1 require further elucidation in future studies. The Morris water maze test was only performed during the fourth-week post‑SAH; therefore, potential changes in the early ability of spatial memory and learning ability may not have been assessed. Finally, IF was used to assess ASK1 co‑localization; however, only 2 animals/experimental group was assessed. The small sample size is a limitation of the present study that may result in inappropriate conclusions and should only be used for qualitative,  rather than quantitative, assessment.

In conclusion, ASK1 inhibitor NQDI‑1 decreased oxidative stress and apoptosis and improved short and long‑term neurological function following SAH via inhibition of ASK1 phosphorylation and the p38 and JNK signaling pathways.  NQDI‑1 may be a potential therapeutic agent for the treatment of  SAH.

Acknowledgments

Not applicable.

Funding

The present study was supported by The National Natural  Science Foundation of China (grant no. 81870944),  The Beijing Science and Technology Plan Subject:  Beijing‑Tianjin‑Hebei Collaborative Innovation Promotion  Project (grant no. Z181100009618035), The National Natural  Science Foundation of China (grant no. 81771233), Beijing  Municipal Administration of Hospitals' Ascent Plan (grant no. DFL20190501) and Research and Promotion Program of  Appropriate Techniques for Intervention of Chinese High‑risk  Stroke People (grant no. GN‑2020R0007).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Authors' contributions

JD, WY, JJ, FL, and AL participated in the experimental design.  JD, JJ, JW, and XY performed the experiments and collected and analyzed the data. XY, FL, and AL interpreted the data. JD and WY drafted the manuscript. XY, FL, and AL revised the manuscript and proofread the language. All authors have read and approved the final manuscript. JD, JW, and FL confirm the authenticity of all the raw data.

Ethics approval and consent to participate

All experimental procedures were approved by the Institutional  Animal Care and Use Committee of Central South University  (approval. no. 2019sydw0104).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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