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To determine the effect of PACA on NGF and proNGF protein levels in mouse brain, NGF and proNGF protein levels were determined by Western blotting using specific antibodies. In mice treated with MPTP, NGF protein levels in SNpc were reduced. PACA effectively restores NGF protein levels to resist the neurotoxicity of MPTP. MPTP greatly increased the accumulation of proNGF, while PACA reduced proNGF protein levels in the midbrain of mice treated with MPTP.
In order to verify the effect of PACA on NGF and proNGF protein levels, we further measured NGF levels through immunofluorescent staining. Midbrain tissue was recovered from mice after treatment with a combination of vehicle, MPTP, MPTP and PACA. MPTP almost eliminated binding of anti-NGF antibodies, while PACA restored retention of anti-NGF antibodies in the midbrain of MPTP-treated mice. Based on immunostaining of NGF and GFAP in the substantia nigra, a dose of 15 mg/kg/day PACA increased NGF levels not only in astrocytes, but also in other cell types. These results suggest that PACA increases the conversion of proNGF to NGF in midbrain tissue.