Isolation and Characterization of NP-POL Nonapeptide for Possible Therapeutic Use in Parkinson's Disease

Abstract

Colostrum and milk are the initial mammalian nourishment and rich reservoir of essential nutrients for newborn development. Bioactive peptides isolated from natural sources, such as colostrum, serve as endogenous regulators and can be used as alternative therapeutic agents in the treatment of neurodegenerative diseases. One example is the previously unknown NP-POL nonapeptide isolated from Colostrinin. In the present study, we investigated a method of NP-POL nonapeptide isolation using Bio-Gel P2 molecular sieve chromatography. We showed the protective effect of NP-POL on 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity using rat adrenal pheochromocytoma (PC12 Tet On) cells. Treatment of PC12 cells with NP-POL nonapeptide reduced 6-OHDA-induced apoptosis and caused transient phosphorylation of extracellular signal-regulated kinases (ERK 1/2), which were shown to promote cell survival. NP-POL nonapeptide also protected neuronal cells against oxidative injury induced by 6-OHDA. These results showed a potential use of NP-POL in the therapy of Parkinson's disease.

Figure 1

Diagram of the NP-POL peptide isolation process.

Figure 2

Separation of the NP-POL peptide from Colostrinin by Bio-Gel P2 molecular sieve beads.

Figure 3

The effects of NP-POL on 6-OHDA-induced cytotoxicity of PC12 cells. PC12 cells (1 × 10⁴/well) were incubated for 24 h with inducers: NP-POL (1 μg/ml–150 μg/ml) (a) or toxin 6-OHDA (1 μM–200 μM) (b). To measure the neuroprotective effect of NP-POL, the nonapeptide was preincubated with cells for 1 h before application of 150 μM 6-OHDA (c) or was applied simultaneously with 6-OHDA (150 μM) (d). Cell viability was measured by the MTT assay. Data are presented as mean ± SD (n = 3). Results were considered significant at ^# p < 0.001 versus untreated cells and at ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 versus 6-OHDA.

Figure 4

The neuroprotective effect of the NP-POL peptide on NGF-differentiated PC12 cell viability treated with 6-OHDA neurotoxin. (a) Control, (b) NP-POL 1 μg/ml, (c) NP-POL 10 μg/ml, (d) NP-POL 100 μg/ml, (e) 6-OHDA 160 μM, (f) NP-POL 1 μg/ml + 6-OHDA 160 μM, (g) NP-POL 10 μg/ml + 6-OHDA 160 μM, and (h) NP-POL 100 μg/ml + 6-OHDA 160 μM. Scale bar = 50 μM.

Figure 5

The effect of the NP-POL peptide on intracellular ROS generation induced by 6-OHDA. (a) NP-POL significantly reduced intracellular ROS levels in PC12 cells. (b) 6-OHDA induced an exponential increase in intracellular ROS in exposed cells. Both 1 h preincubation (c) with selected doses of NP-POL before 24 h of 6-OHDA exposure and 24 h of coincubation with NP-POL and 6-OHDA (d) resulted in a significant decrease in intracellular ROS levels. Data are expressed as mean ± SD (n = 3). Results were considered significant at ^# p < 0.001 versus untreated cells and ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 versus 6-OHDA.

Figure 6

The effect of NP-POL peptide on ERK 1/2 kinase activity. (a) NP-POL transiently enhanced ERK 1/2 activation at the selected dose of 100 μg/ml after 5 min of incubation. (b) Stable 6-OHDA-enhanced ERK 1/2 activation after 60 min of incubation. (c) Coincubation of NP-POL with 6-OHDA resulted in the transient activation of ERK 1/2 after 5 and 15 min. Data are presented as mean ± SD (n = 3). Results were considered significant at ^∗ p < 0.05 and ^∗∗∗ p < 0.001 versus untreated cells.