A newly-synthesized compound CP-07 alleviates microglia-mediated neuroinflammation and ischemic brain injury via inhibiting STAT3 phosphorylation

Mengdi Guo; Qian Cao; Shengnan Xia; Xiang Cao; Jian Chen; Yi Qian; Xinyu Bao; Yun Xu

doi:10.2478/jtim-2023-0090

A newly-synthesized compound CP-07 alleviates microglia-mediated neuroinflammation and ischemic brain injury via inhibiting STAT3 phosphorylation

J Transl Int Med. 2023 Jul 5;11(2):156-168. doi: 10.2478/jtim-2023-0090. eCollection 2023 Jul.

Authors

Mengdi Guo^{1

2

3

4}, Qian Cao⁵, Shengnan Xia^{1

2

3

4}, Xiang Cao^{1

2

3

4}, Jian Chen^{1

2

3

4}, Yi Qian^{1

2

3

4}, Xinyu Bao^{1

2

3

4}, Yun Xu^{1

2

3

4

5}

Affiliations

¹ Department of Neurology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, Jiangsu Province, China.
² Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University, Nanjing 210008, Jiangsu Province, China.
³ Jiangsu Province Stroke Center for Diagnosis and Therapy, Nanjing 210008, Jiangsu Province, China.
⁴ Nanjing Neuropsychiatry Clinic Medical Center, Nanjing 210008, Jiangsu Province, China.
⁵ Nanjing Drum Tower Hospital Clinical College of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210008, Jiangsu Province, China.

Abstract

Background and objectives: Overactivated glial cells, especially microglia, are core components in the progression of pathologic neuroinflammation, and the application of anti-inflammatory reagents has been regarded as a potential therapy in the management of infarction/reperfusion (I/R) brain injury. This research aims to clarify the anti-inflammatory efect of a novel lipophilic compound N-(2-[4-tert-butylphenyl]-2-[pyrrolidine-1-yl]ethyl)-7-methyl-4-oxo-4H-chromene-2-carboxamide (named CP-07 in this study) in LPS-stimulated BV2 cell line and primary mouse microglia, and its therapeutic effect on I/R brain injury.

Method: Cell Counting Kit-8 assay was used to determine the maximal nontoxic dose of CP-07. The mRNA levels of representative proinflammatory cytokines were determined by quantitative real-time polymerase chain reaction both in vitro and in vivo. TTC staining was performed to calculate infarct volumes while behavioral tests were used to assess the neurological deficits at 24 h after middle cerebral artery occlusion (MCAO). Flow cytometry analysis and immunofluorescence staining were performed to calculate the percentage of pro-inflammatory microglia in vivo.A selective JAK2/STAT3 pathway inhibitor, AG490 was used to block STAT3 phosphorylation before the CP-07 anti-inflammation tests in vitro.

Results: CP-07 could effectively suppress the mRNA levels of IL-6, IL-1β, iNOS and TNF-α induced by lipopolysaccharide (LPS) in vitro, and markedly block the evaluation of the fluorescence intensity of Iba-1 in primary mouse microglia. In middle cerebral arteryocclusion models, intraperitoneal injection with 1 mg/kg CP-07 significantly reduced cerebral infarct volumes at 24 h after surgery compared with vehicle treatment group, and promoted the recovery of neurological functions in MCAO mice. Further studies validated that CP-07 administration reduced the percentage of CD86 positive microglia after I/R injury, and the expression level of p-STAT3 was also markedly reduced in both microglial cells and the penumbra tissues. Blocking STAT3 phosphorylation with AG490 could completely eliminate the anti-inflammatory effects of CP-07, at least in vitro.

Conclusion: We showed that a newly synthesized compound, CP-07, could effectively reduce the inflammatory responses in LPS-stimulated BV2 cells and primary mouse microglia, and overproduction of cytokines in middle cerebral artery occlusion mouse models by inhibiting STAT3 phosphorylation, leading to a neuroprotective effect on I/R brain injury.

Keywords: Ischemic stroke; STAT3; anti-inflammatory therapy; microglia; neuroinflammation.

Grants and funding

This research was supported by the National Natural Science Foundation of China (NO. 81920108017, NO. 82130036 and NO. 81630028), the Key Research and Development Program of Jiangsu Province of China (BE2020620), and Jiangsu Province Key Medical Discipline.