J Vet Sci.  2018 Jul;19(4):512-518. 10.4142/jvs.2018.19.4.512.

Sciatic nerve leachate of cattle causes neuronal differentiation of PC12 cells via ERK1/2 signaling pathway

• Zhang Z
• Liu Y
• Zhu X
• Wei L
• Zhu J
• Shi K
• Wang G
• Pan L

• KMID: 2417566
• DOI: http://doi.org/10.4142/jvs.2018.19.4.512

Abstract

Previous studies have shown that the sciatic nerve has neurotrophic activity, and nerve regeneration, differentiation, and axon outgrowth can be modulated by different sciatic nerve preparations. However, numerous animals may have to be sacrificed to obtain enough sciatic nerves to make a sciatic nerve preparation. Some studies have demonstrated that the role of sciatic nerve preparations in neural differentiation depends on the neurotrophins that Schwann cells secrete, and these factors are highly conserved among different species. To reduce the use of experimental animals, in this study, we made a leachate by using the sciatic nerve of cattle and explored its effect on neuronal differentiation of rat PC12 cells (a useful model for studying neuronal differentiation). Results showed the neurite outgrowth of PC12 cells treated with the cattle sciatic nerve leachate for 3, 6, and 9 days was significantly improved, and the expressions of β3-tubulin and microtubule-associated protein 2 (two neuron-specific proteins) were increased. Moreover, the ERK1/2 signaling pathway was activated after PC12 cells were incubated with cattle sciatic nerve leachate for 9 days. Thus, a sciatic nerve leachate obtained from cattle can effectively induce neuronal differentiation of rat PC12 cells via ERK1/2 signaling pathway.

Keyword

PC12 cells; Schwann cells; sciatic nerve

MeSH Terms

Animals
Axons
Cattle*
Microtubule-Associated Proteins
Nerve Growth Factors
Nerve Regeneration
Neurites
Neurons*
PC12 Cells*
Rats
Schwann Cells
Sciatic Nerve*
Microtubule-Associated Proteins
Nerve Growth Factors

Figure

• 

  Fig. 1 Cattle sciatic nerve leachate induces neurite outgrowth of PC12 cells. The number of PC12 cells with neurites that was treated with or without sciatic nerve leachate and 10 µM PD98059 for 3, 6, and 9 days were analyzed. Data are expressed as mean ± SEM values. ap < 0.05 compared with control; bp < 0.05 compared with sciatic nerve leachate alone group. One-way ANOVA and Duncan's multiple comparison tests were used.

• 

  Fig. 2 Expressions of β3-tubulin and MAP2 proteins in PC12 cells were observed by immunofluorescence assay. Expressions of β3-tubulin and MAP2 proteins in PC12 cells incubated with or without cattle sciatic nerve leachate or 10 µM PD98059 for 9 days were observed under immunofluorescence microscopy. Compared to the control group, expression of β3-tubulin and MAP2 proteins were increased in PC12 cells treated with sciatic nerve leachate. Moreover, 10 µM of PD98059 effectively alleviated sciatic nerve leachate-induced over-expressions of β3-tubulin and MAP2 proteins. Scale bars = 20 µm.

• 

  Fig. 3 Expression levels of β3-tubulin and MAP2 proteins in PC12 cells were examined by Western blotting. (A) The bands of β3-tubulin and MAP2 proteins in PC12 cells incubated with or without sciatic nerve leachate (SNL) or 10 µM PD98059 for 9 days were observed by Western blotting. (B) Protein levels of β3-tubulin and MAP2 were measured by densitometry. Real-time values were normalized to β-actin. Data are expressed as mean ± SEM values. ap < 0.05 compared with control; bp < 0.05 compared with SNL alone group. One-way ANOVA and Duncan's multiple comparison tests were used.

• 

  Fig. 4 Effects of cattle sciatic nerve leachate (SNL) on ERK1/2 phosphorylation. (A) The bands of p-ERK1/2 and ERK1/2 proteins in PC12 cells incubated with or without SNL or 10 µM PD98059 for 9 days were observed by Western blotting. (B) Protein levels of ERK1/2 were measured by densitometry. Real-time values were normalized to β-actin. Data are expressed as mean ± SEM values. (C) Protein levels of p-ERK1/2 were measured by densitometry. Real-time values were normalized to β-actin. Data are expressed as mean ± SEM values. ap < 0.05 compared with control; bp < 0.05 compared with SNL alone group. One-way ANOVA and Duncan's multiple comparison tests were used.

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