Ischemic stroke is a high mortality disease that causes irreversible damage. Chlorogenic acid is a polyphenolic substance with neuroprotective properties. Bcl-2 family proteins perform a critical role in apoptosis process. Bcl-2 and Bcl-xL are anti-apoptotic proteins that prevent cell death, and Bax and Bad are pro-apoptotic proteins that promote apoptosis. We investigated whether chlorogenic acid modulates Bcl-2 family proteins during focal cerebral ischemia. We made a rat model of ischemic stroke by performing middle cerebral artery occlusion (MCAO). Chlorogenic acid (30 mg/kg) or phosphate-buffered saline was treated via intraperitoneal injection 2 hr before MCAO. Neurological behavioral tests were performed 24 hr after MCAO damage and cortical tissues were collected. Reverse transcription-PCR, Western blot, and immunofluorescence staining were performed to observe changes in Bcl-2 family proteins expression. MCAO-damage induced neurobehavioral disorders and chlorogenic acid alleviate these deficits. Bcl-2 and Bcl-xL expressions were decreased and Bax and Bad expressions were increased in MCAO animals. However, chlorogenic acid treatment attenuated the decrease of Bcl-2 and Bcl-xL and the increase of Bad and Bax due to MCAO surgery. Moreover, chlorogenic acid treatment attenuated MCAO-induced upregulation of caspase-3. These findings suggest that chlorogenic acid exerts neuroprotective effects against MCAO damage by regulating Bcl-2 family proteins including Bcl-2, Bcl-xL, Bax, and Bad.

Introduction
Materials and Methods
    1. Experimental animals and drug treatment
    2. Middle cerebral artery occlusion (MCAO)
    3. Neurological deficit scoring test
    4. Corner test
    5. Reverse transcription-polymerase chain reaction(RT-PCR)
    6. Western blot analysis
    7. Immunofluorescence staining
    8. Statistical analysis
Results and Discussion
    1. Neuroprotective effect of chlorogenic acid inMCAO animals
    2. Regulation mRNA expression of Bcl-2 familyproteins by chlorogenic acid in MCAO animals
    3. Regulation of Bcl-2 family proteins by chlorogenicacid in MCAO animals
    4. Modulation of caspase-3 protein expression bychlorogenic acid in MCAO animals
Acknowledgements
References

• Ju Bin Kang(Graduate Student, Department of Veterinary Anatomy and Histology, College of Veterinary Medicine, Research Institute of Life Science, Gyeongsang National University, Jinju-daero 501, Jinju 52828, Korea)
• Myeong Ok Kim(Professor, Division of Life Science and Applied Life Science, College of Natural Sciences, Gyeongsang National University, jinju-daero 501, Jinju 52828, Korea) Corresponding author
• Phil Ok Koh(Professor, Department of Veterinary Anatomy and Histology, College of Veterinary Medicine, Research Institute of Life Science, Gyeongsang National University, Jinju-daero 501, Jinju 52828, Korea) Corresponding author