p66shc siRNA-Encapsulated PLGA Nanoparticles Ameliorate Neuropathic Pain Following Spinal Nerve Ligation

Abstract

p66shc, a member of the shc adaptor protein family, has been shown to participate in regulation of mitochondrial homeostasis, apoptosis, and autophagosome formation. The present study was performed to investigate whether p66shc siRNA-encapsulated poly(d,l-lactic-co-glycolic acid) nanoparticles (p66shc siRNA-PLGA NPs) can attenuate spinal nerve ligation (SNL)-induced neuropathic pain in rats. The SNL-induced pain behavior was decreased in the p66shc siRNA-PLGA NP-treated group compared with the scrambled siRNA-PLGA NP-treated group. In the L5 spinal cord of the p66shc siRNA-PLGA NP-treated group, expression levels of phosphorylated p66shc, cleaved caspase-3, p62, and PINK1, as well as microglial activation, were also decreased. In addition, p66shc knockdown using p66shc siRNA reduced the expression levels of cleaved caspase-3, p62, and PINK1, as well as proinflammatory mediators in the H₂O₂-treated HT22 neuronal cells. These results suggest that downregulation of p66shc expression in the spinal cord using p66shc siRNA-PLGA NPs could reduce the SNL-induced neuropathic pain by attenuating the SNL-induced aberrant autophagic, mitophagic, and neuroinflammatory processes in rats.

Keywords: PLGA nanoparticle; autophagy; neuropathic pain; p66shc; proinflammatory mediators; spinal nerve ligation.

Figure 1

Characterization of p66shc siRNA-PLGA NPs. p66shc siRNA-PLGA NPs were prepared by briefly sonicating the PLGA mixture and then examined for (A) size (d.nm: diameter in nm); (B) zeta potential using the Zetasizer Nano ZS90; (C) by scanning electron microscopy; scale bar = 200 nm, and (D) the cumulative siRNA release from PLGA NPs.

Figure 2

The degree of pain confirmed by the mechanical threshold after spinal nerve ligation (SNL). (A) experimental timeline of this study; (B) After three days of SNL-induced neuropathic pain, the mechanical threshold was identified using the von Frey filament test to confirm the analgesic effect of p66shc siRNA-PLGA NPs (n = 6 in each group). Data are expressed as means ± SEM (two-way ANOVA with Dunnett’s post hoc test, *** p < 0.001 vs. sham group, ^### p < 0.001, ^## p < 0.01 vs. SNL group.

Figure 3

p66shc siRNA-PLGA NPs reduced the protein levels of cleaved caspase-3, p62, and PINK1 in the ipsilateral spinal cord at 7 days after SNL. (A,B) phosphorylation of p66shc was decreased after treatment with p66shc siRNA-PLGA NPs; (C,D) SNL-induced increases in cleaved caspase-3, p62, and PINK1 protein levels were significantly attenuated in the p66shc siRNA-SNL group. β-actin was used as a loading control. Data are expressed as means ± SEM (unpaired Student’s t-test, *** p < 0.001, ** p < 0.01, * p < 0.05 vs. sham or SNL group).

Figure 4

Iba-1 immunoreactivity in the ipsilateral spinal cord at seven days after SNL. (A) microglial activation was reduced in the p66shc siRNA-PLGA NPs group compared with the SNL group. Scale bar = 100 μm; (B) intensity of Iba-1 immunoreactivity in the ipsilateral spinal cord after SNL. Data are expressed as means ± SEM (unpaired Student’s t-test, *** p < 0.001 vs. sham, * p < 0.05 vs. SNL).

Figure 5

Reduced expression of markers of apoptosis, autophagy, and mitophagy after p66shc knockdown in H₂O₂-induced HT22 cells. (A) induction of oxidative stress by H₂O₂ after p66shc knockdown in the HT22 neuronal cell line reduced the protein levels of phosphorylated p66shc, cleaved caspase-3, p62, and PINK1; (B) β-actin was used as a loading control. Western blotting showed that the levels of p66shc, cleaved caspase-3, p62, and PINK1 were significantly altered in HT22 cells under oxidative stress induced by H₂O₂. Data are expressed as means ± SEM (unpaired Student’s t-test, *** p < 0.001, ** p < 0.01).

Figure 6

p66shc knockdown resulted in decreased mRNA levels of proinflammatory mediators in H₂O₂-induced HT22 cells. TNF-α: tumor necrosis factor-α, IL-1β: interleukin-1β, IL-6: interleukin-6, COX-2: cyclooxygenase-2, iNOS: inducible nitric oxide synthase. Data are expressed as means ± SEM (unpaired Student’s t-test, *** p < 0.001, ** p < 0.01, * p < 0.05).